Oligosaccharide modification and labeling of proteins

ABSTRACT

The present invention generally relates to methods of functionalizing proteins, particularly antibodies, at oligosaccharide linkages, methods of humanizing antibodies by modifying glycosylation, as well as to novel antibodies linked to modified oligosaccharides. The invention further relates to kits that may be used to produce the antibodies of the invention.

This application is a Divisional application of U.S. Non Provisionalapplication Ser. No. 14/221,798, filed Mar. 21, 2014, which is aDivisional application of U.S. Non Provisional application Ser. No.11/674,136, filed Feb. 12, 2007, which claims the benefit of U.S.Provisional Application No. 60/772,221, filed Feb. 10, 2006 and U.S.Provisional Application No. 60/804,640, filed Jun. 13, 2006, thecontents of which are incorporated by reference as if set forth fullyherein.

FIELD OF THE INVENTION

The invention generally relates to methods of labeling proteins at noveloligosaccharide linkages, methods of humanizing antibodies by modifyingglycosylation, as well as to novel antibodies linked to modifiedoligosaccharides.

BACKGROUND

Isolated or synthesized proteins and antibodies, such as IgGs, are usedtherapeutically and for diagnostic and research purposes. By labelingantibodies with detectable labels, such as, for example, fluorophores,antibodies can be used to specifically detect target biologicalmolecules or cells. Antibodies may also be tagged with binding reagents,such as, for example, biotin, so that they may be used to specificallybind target biological molecules or cells, followed by purification ofthe biological molecule or cell by using a reagent that binds to thetagged antibody, for example, streptavidin. Antibodies have generallybeen labeled at cysteine or lysine residues, which may often be presentin the Fab, or binding portion of the antibody. Adding tags or labels inthis region may disrupt or at least alter the binding properties of theantibody. Further, it is often difficult to quantitate the number oflabeled molecules attached to each antibody.

Therapeutic monoclonal antibodies (Mabs) have become indispensable drugsto combat cancer, rheumatoid arthritis, macular degeneration, and otherdiseases or conditions. However, antibodies generated in non-human celllines may have antigenic features recognized as foreign by the humanimmune system, limiting the antibodies' half-life and efficacy.Incorporating human IgG sequences into transgenic mice has reduced, butnot eliminated, immunogenicity problems. Besides the protein sequence,the nature of the oligosaccharides attached to the IgG has a profoundeffect on immune-system recognition. Because glycosylation is cell typespecific, IgGs produced in different host cells contain differentpatterns of oligosaccharides, which could affect the biologicalfunctions. Even where cells, such as human embryonic stem cells, aregrown on mouse feeder layers in the presence of animal-derived serumreplacements, the cells incorporated a nonhuman, and immunogenic, sialicacid, and the sialic acid was then found on the cell surface. (Martin,M. J., et al., Nature Medicine, 2005, 11:228-232). Although thetherapeutic antibody industry has tried to avoid these problems byproducing less antigenic IgG with defucosylated oligosaccharides,defucosylated antibodies are not equivalent to humanized antibodies, andmay still have immunogenecity issues, as well as having differenthalf-lives than natural human antibodies.

Metabolic oligosaccharide engineering refers to the introduction ofsubtle modifications into monosaccharide residues within cellularglycans. Researchers have used metabolic engineering to disrupt glycanbiosynthesis, chemically modify cell surfaces, probe metabolic fluxinside cells, and to identify specific glycoprotein subtypes from theproteome. (reviewed in Dube, D. H., and Bertozzi, C. R., Current Opinionin Chemical Biology, 2003, 7:616-625).

There is a need for antibodies that have tags or labels at sites otherthan the binding region, and for antibodies that may be easily labeledusing simple and efficient chemical reactions. There is also a need forantibodies that have post-translational modifications that are more likehuman antibodies.

SUMMARY OF THE INVENTION

The invention generally relates to methods of remodeling and labelingproteins and antibodies at novel oligosaccharide linkages, methods ofhumanizing antibodies by modifying glycosylation, as well as toantibodies or proteins linked to modified oligosaccharides. Theantibodies or proteins, for example, IgGs, are labeled using either invitro or in vivo methods.

In some in vitro embodiments, an oligosaccharide present on a firstantibody is cleaved, and a different, second, oligosaccharide isattached to the cleavage site on the first antibody. This secondoligosaccharide may be, for example, cleaved from a second antibody.Using this method, oligosaccharides obtained from, for example, humanantibodies may be attached to antibodies generated in non-human celllines. Also, using this method, secondary labels may be attached to theantibodies at an oligosaccharide.

In some in vivo embodiments, unnatural sugars having chemical handlesare taken up by antibody-producing cells, and incorporated into theoligosaccharides on the antibodies. Once the antibodies are isolated,secondary labels may be attached at the chemical handles.

By labeling antibodies at glycan residues in the Fc portion of theantibody, instead of the traditional labeling of cysteine or lysineresidues in the binding region of the antibody, possible disruption ofepitope binding is avoided. In addition, the number of glycan residuespresent on the IgG, is generally known. In contrast, eachepitope-specific IgG may have a different number of lysine or cysteineresidues in the peptide sequence, and each may have a different abilityto be labeled, based on the IgG structure. Therefore, it would generallybe difficult to determine how many labels were present on each IgG.Using the methods of the present invention, it is expected that aboutall of the glycan residues are labeled, which allows for quantitativelabeling and detection of the antibodies using, for example,fluorescence-activated cell sorting (FACS).

In other methods of the invention, the ligation of humanoligosaccharides to non-human IgG would result in glycoforms practicallyidentical to those in humans, with the only difference being an extragalactose near the sugar attachment point, and a ring structure from thecycloaddition.

Besides the humanization of therapeutic antibodies, there are manypossible applications for the methods of the present invention. Forexample, glycosylation patterns can alter in certain diseases orconditions such as, for example, rheumatoid arthritis and pregnancy. Theability to mix and match oligosaccharides may enable researchers toinvestigate human diseases involving altered glycosylation in animalmodels.

One aspect of the invention provides a method of producing aglycomodified protein, comprising:

-   -   attaching a modified sugar comprising a chemical handle to a        GlcNAc residue on a first protein; and    -   mixing said first protein with a reporter molecule, carrier        molecule or solid support capable of reacting with said chemical        handle;    -   wherein said reporter molecule, carrier molecule or solid        support attaches to the protein at said chemical handle, thereby        forming a glycomodified protein.

In another embodiment, said first protein is an antibody. In a moreparticular embodiment, the antibody is IgG.

In another embodiment, the attaching step is in a solution substantiallyfree of proteases.

In another embodiment, prior to the attaching step, the method comprisescleaving an oligosaccharide present on a first protein at aGlcNAc-GlcNAc linkage to obtain a protein comprising a GlcNAc residue.In a more particular embodiment, said oligosaccharide is cleaved usingendoglycosidase H cleavage at the GlcNAc-GlcNAc linkage. In anotherembodiment, said oligosaccharide is cleaved using endoglycosidase Mcleavage at the GlcNAc-GlcNAc linkage.

In another embodiment, said reporter molecule, carrier molecule or solidsupport is attached to said GlcNAc using a mutant galactosyltransferase. In another embodiment, said mutant is a Y289L mutant.

In another embodiment, said modified sugar is an azide-modified sugarand said reporter molecule, carrier molecule or solid support is labeledwith alkyne or activated alkyne. More particularly, said azide-modifiedsugar is UDP-GalNAz.

In another embodiment, prior to the attaching step, the methodcomprises:

-   -   cleaving an oligosaccharide present on a second protein at a        GlcNAc-GlcNAc linkage to obtain an oligosaccharide having a        GlcNAc residue;    -   binding said oligosaccharide to the reporter molecule, solid        support or carrier molecule capable of reacting with said        chemical handle.

In another embodiment, the oligosaccharide having a GlcNAc residue istreated with ammonium bicarbonate and said oligosaccharide is attachedto an alkyne by a succinimidyl ester. In another embodiment, said secondprotein is synthesized in a different cell line or cell type than saidfirst protein. In another embodiment, said second protein is synthesizedin a human cell.

In another embodiment, the reporter molecule is selected from the groupconsisting of a fluorescent dye, an enzyme, a radiolabel, a metalchelator, or a detectable substrate. In another embodiment, the carriermolecule is selected from the group consisting of therapeutic agents,DNA, protein, peptides, and sugars.

In another embodiment, the glycomodified protein becomes more or lessantigenic as compared to the first protein before it is modified. Inanother embodiment, the first protein is derived from a non-humansource. In another embodiment, the glycomodified protein is humanized

In another embodiment, said cleavage of said first protein is performedin the presence of an OH-alkyne, and said endoglycosidase M attachessaid OH-alkyne to the cleaved GlcNAc residue; and said modifiedoligosaccharide is labeled with an azide residue.

Another aspect of the invention provides a method of labeling an proteinby labeling an oligosaccharide attached to said protein, comprisingincubating an protein-producing cell in the presence of an unnaturalsugar, wherein said unnatural sugar comprises a chemical handle.

Another aspect of the invention provides an antibody comprising alabeled oligosaccharide.

Irrespective of their location, the embodiments are provided as moreparticular descriptions of any one of the aspects of the invention.

Other objects, features and advantages of the present invention willbecome apparent from the detailed description. It should be understood,however, that the detailed description and the specific examples, whileindicating preferred embodiments of the invention, are given by way ofillustration only, since various changes and modifications within thespirit and scope of the invention will become apparent to those skilledin the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a comparison of undigested (lower panel) andEndoH_(f)-treated (top panel) goat anti-rabbit polyclonal antibody byHPLC. For undigested antibody the light chain eluted at 20.2 minutes andthe glycosylated heavy chain at 26.8 minutes. For EndoH_(f)-treatedantibody, the fully deglycosylated heavy chain eluted at 31.2 minutes.

FIG. 2 shows a comparison of undigested (lower panel) andEndoH_(f)-treated (top panel) G2A monoclonal antibody analyzed by HPLC.For undigested antibody the light chain eluted at 21.5 minutes andglycosylated heavy chain at 29.1 minutes. For EndoH_(f)-treatedantibody, the light chain eluted at 21.1 minutes, glycosylated orpartially deglycosylated heavy chain at 27.8 and 28.7 minutes, and fullydeglycosylated heavy chain at 30.1 minutes.

FIG. 3A shows TAMRA Click-iT™ label, showing labeling of chickenanti-goat heavy chain in Endo H-treated IgG (Degly), but not untreatedIgG (Undegly). C: Control chicken anti-goat IgG starting material;Degly: Chicken anti-goat IgG after Endo H digestion, O-GlcNAc enzymaticlabeling and Click iT detection; Undegly: Chicken anti-goat IgG afterO-GlcNAc enzymatic labeling and Click iT detection. FIG. 3B shows thesame gel described in FIG. 3A post-stained with SYPRO Ruby gel stain toshow total protein pattern.

FIG. 4A shows M96 cells fed DMSO vehicle (control), or 20 μM Ac₄GalNAz,40 μM Ac₄GalNAz, 20 μM Ac₄ManNAz, 40 μM Ac₄ManNAz, or 30 μM Ac₄GlcNAz,respectively, followed by Click iT detection of incorporated azidosugars.

FIG. 4B shows the same gel described in FIG. 4A post-stained with SYPRORuby gel stain to show total protein pattern.

FIGS. 5A, 5B and 5C show metabolic labeling and “click” detection ofglycoprotein subclasses: FIG. 5A and FIG. 5B show detection afterseparation on a gel and FIG. 5C shows detection schematically.

FIG. 6 shows soluble Jurkat cell proteins that had been fed Ac₄GlcNAz orDMSO vehicle (control unfed)—followed by Click iT detection ofincorporated azido sugars and separation by 2-D polyacrylamide gelelectrophoresis. The same gels were post-stained with SYPRO Ruby gelstain to show total protein pattern.

FIG. 7 shows in gel detection of 40 and 50 kD azide-labeled modelproteins, which were first labeled with a fluorescent alkyne tag andthen separated on the gel.

FIG. 8 shows that labeling efficiency of 40 and 50 kd azide-labeledmodel proteins is unchanged in complex protein extracts.

FIG. 9A shows the GalT1 enzymatic labeling and detection of α-crystallinO-GlcNAc schematically and FIG. 9B shows detection after separation on agel.

FIG. 10A shows a comparison of GalT1 enzymatic labeling and Click-iTdetection of α-crystallin O-GlcNAc to FIG. 10B which shows detectionwith the O-GlcNAc Monoclonal Antibody CTD 110.6.

FIG. 11 shows the multiplex detection of O-GlcNAc modified proteins,phosphoproteins and total proteins in the same 2-D gel.

FIG. 12 shows multiplexed western bot detection of O-GlcNAc modifiedproteins and cofilin on the same membrane.

FIG. 13 shows the differential detection of O-GlcNAc modified proteinsin control and O-GlcNAcase inhibitor(PUGNAc)-treated cultured cellextracts.

FIGS. 14A and 14B depict gels showing the results of separating proteinslabeled using the click reaction with different chelators. 2.5 ng eachof azido-ovalbumin and azido-myoglobin spiked into 80 ug of unlabeledJurkat lysate was labeled with TAMRA alkyne for 2 hrs. The reactioncontained 50 mM TRIS pH8, 25% propylene glycol, 1mM CuSO₄, 5 mM sodiumascorbate, 20 uM TAMRA alkyne. The reactions were performed with andwithout chelator (10 mM TPEN [upper left gel], EDTA [upper right gel],bathocuproine disulfonic acid (BCS) [middle left gel] or neocuproine[middle right gel]). Control reactions were performed without CuSO₄[lower left gel] or without chelator [lower right gel]. After labeling,the samples were precipitated, resolubilized in 7 mM urea/2mMthiourea/65 mM DTT/2% CHAPS/ and approximately 30 μg was analyzed on 2-Dgels (pH 4-7 IEF strips, 4-12% BIS-TRIS gels with MOPS buffer). TheTAMRA signal was imaged at 532 nm excitation, 580 long pass emission ona Fuji FLA3000 (FIG. 14A) then the gels were post-stained with Sypro®Ruby total protein gel stain (FIG. 14B).

FIGS. 15A and 15B depict gels showing the results of separating proteinslabeled using the click reaction with different chelators. The samplesand click labeling conditions are the same as for FIGS. 14A and 14B,except that chelator treatments include addition of either 5 mM TPEN,BCS or Neocuproine at the beginning of the reaction. After labeling, thesamples were precipitated, resolubilized in LDS buffer+5 mM TCEP andserial 2-fold dilutions were performed. Dilutions were loaded onto 4-12%BIS-TRIS gels with MOPS running buffer (250 ng each of ovalbumin andmyoglobin in lane 1). FIG. 15A shows that the chelators reduce thebackground of the image for the TAMRA signal without compromisingsensitivity. In FIG. 15B, post-staining with Sypro® Ruby total proteingel stain shows that the band resolution is much better for the sampleswith chelator.

FIGS. 16A and 16B: The samples and click labeling conditions are thesame as for FIGS. 14A and 14B, except that chelator treatments includeaddition of either 7 mM, 5 mM or 2 mM BCS or neocuproine. The lanesmarked with an asterisk in FIG. 16A indicate reactions in which theCuSO₄ and BCS were added to the reaction and vortexed prior to addingthe sodium ascorbate. In all other reactions the CuSO₄ and sodiumascorbate were added and vortexed prior to adding the BCS. The gels showthat it is imperative to add the sodium ascorbate and CuSO₄ to thereaction tube and mix prior to adding the chelator. If the chelator andCuSO₄ are added and vortexed prior to adding the sodium ascorbate, theazide-alkyne labeling does not proceed, suggesting that the chelatorinhibits the reduction of Cu II to Cu I.

FIGS. 17A and 17B show the enzymatic labeling of antibodies using clickchemistry: FIG. 17A) Lane 1—Goat antibody only (GAb); Lane 2—GAb withGalT1 enzyme but without UDP-GalNAz Control; Lane 3—GAb with enzyme andUDP-GalNAz. Lane 4—azide-labeled ovalbumin and myoglobin controlproteins; Lane 5—MW markers unlabeled and FIG. 17B) Azide-labeled goatantibodies (from above) were run as a dilution series followed bypost-staining with SYPRO Ruby gel stain to show total protein pattern.

FIGS. 18A, 18B, 18C, 18D, 18E and 18F show structures of four present(FIGS. 18A-18D) and two potential (FIGS. 18E,18F) alkyne fluorophoresthat can be used to label biomolecules using the methods of theinvention: FIG. 18A) TAMRA-alkyne; FIG. 18B) Alexa 488-Alkyne; FIG. 18C)Alexa 633-Alkyne; FIG. 18D) Alexa 532-Alkyne, FIG. 18E) a potentialfluorogenic alkyne; FIG. 18F) a potential fluorogenic alkyne.

FIG. 19 shows the results of in-gel staining using the TAMRA-alkynecompound shown in FIG. 12A. Lanes 2, 3, and 4 on the left side of thegel represent cellular extracts that were labeled with azide-modifiedsugars: lane 1 is the control, non-labeled cells. On the right, controlazide labeled proteins (ovalbumin and myoglobin) (+) or non-labeledcontrols (−) are shown at varying concentrations. The results show veryefficient and selective in-gel labeling of azido-modified proteins.

DETAILED DESCRIPTION OF THE INVENTION Introduction:

Various glycosidase enzymes, such as Endo-H are able to cleave N-linkedoligosaccharides and other enzymes, such as Gal-T are able to transferselect oligoaccharides to an acceptor molecule containing an —OH group.The most efficient acceptor molecules are N-acetylglucosamine andglucose. Alternatively, select sugars can be introduced to an acceptor,such as a protein through metabolic labeling. The use of this enzyme ormetabolic system in several applications that involve decoration ofproteins with oligosaccharides (e.g. therapeutic antibodies) or theconjugation of proteins to solid substrates with oligosaccharides isdescribed herein. Some applications may include the transfer ofoligosaccharides to protein containing complementary chemical handlesfor solid support surfaces coated with appropriate acceptor molecules.Additionally, sugars could be transferred to a detection molecule suchas a fluorescent or fluorogenic compound that would enhance detection,purification, and characterization or proteins by mass spectrometry.Transfer to a macromolecule containing an appropriate acceptor OH groupplus an affinity peptide tag, reactive chemical moiety (e.g. azide,alkyne), or a biotin is also possible.

One preferred application of this methodology is the humanization ofnon-human antibodies by replacing the non-human N-linkedoligosaccharides with human N-linked oligosaccharides derived from humanantibodies. In this case, the non-human antibody is decorated with a setof heterogeneous human oligosaccharides. Alternatively, a homogenouspurified or synthetic human-type oligosaccharide, attached to a proteinor peptide substrate, could be used as a donor to impart specificproperties to the antibody acceptor. Such properties might include theregulation of serum half-life, targeting to specific cell or tissuese.g. Fc receptors, or alteration of antibody stability. The donor couldalso contain a functional moiety such as a metal chelator, a fluorescentmolecule, an antigen, an oligonucleotide, a biotin compound, or acellular ligand.

Definitions:

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositionsor process steps, as such may vary. It must be noted that, as used inthis specification and the appended claims, the singular form “a”, “an”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a ligand” includes aplurality of ligands and reference to “an antibody” includes a pluralityof antibodies and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. The following terms aredefined for purposes of the invention as described herein.

TABLE 1 List of Abbreviations Abbreviation Term. GalNAzN-alpha-azidoacetylgalactosamine. GlcNAz N-alpha-azidoacetylglucosamine.GalNAc N-acetylgalactosamine. GlcNAc N-acetylglucosamine LOSLipooligosaccharide. ManLev N-levulinoylmannosamine. ManNAcN-acetylmannosamine. ManNAz N-alpha-azidoacetylmannosamine. ManNButN-butanoylmannosamine. ManNProp N-propanoylmannosamine. NCAM neural celladhesion molecule. PSA Polysialic acid. Endo H Endoglycosidase H Endo MEndoglycosidase M

The term “acceptor” as used herein refers to a substituent capable offorming an endoglycosidase catalyzed bond with an oligosaccharide ormonosaccharide comprising a donor. Preferably, the acceptor unit is aGlcNAc residue appended to an antibody. More preferably, the acceptorunit is a GlcNAc that is targeted by Endo-M or Endo-A and is bound to anadditional saccharide moiety prior to contact with Endo-M or Endo-A.

The term “donor” as used herein refers to a substituent capable offorming an endoglycosidase catalyzed bond with an oligosaccharide ormonosaccharide acceptor. Preferably, the donor unit is a GlcNAc residueappended to an asparagine. More preferably, the donor unit is a GlcNActhat is targeted Endo-M or Endo-A and is bound to an additional GlcNAcmoiety prior to contact with Endo-M or Endo-A.

Preferred donors comprise the structure: Asn-GlcNAc-X, wherein X is theoligosaccharide or monosaccharide that is transferred to the targetprotein comprising an acceptor.

The term “activated alkyne ,” as used herein, refers to a chemicalmoiety that selectively reacts with an alkyne modified group on anothermolecule to form a covalent chemical bond between the alkyne modifiedgroup and the alkyne reactive group. Examples of alkyne-reactive groupsinclude azides. “Alkyne-reactive” can also refer to a molecule thatcontains a chemical moiety that selectively reacts with an alkyne group.

The term “affinity,” as used herein, refers to the strength of thebinding interaction of two molecules, such as an antibody and an antigenor a positively charged moiety and a negatively charged moiety. Forbivalent molecules such as antibodies, affinity is typically defined asthe binding strength of one binding domain for the antigen, e.g. one Fabfragment for the antigen. The binding strength of both binding domainstogether for the antigen is referred to as “avidity”. As used herein“High affinity” refers to a ligand that binds to an antibody having anaffinity constant (K_(a)) greater than 10⁴ M⁻¹, typically 10⁵-10¹¹ M⁻¹;as determined by inhibition ELISA or an equivalent affinity determinedby comparable techniques such as, for example, Scatchard plots or usingK_(d)/dissociation constant, which is the reciprocal of the K_(a).

The term “alkyne reactive,” as used herein, refers to a chemical moietythat selectively reacts with an alkyne modified group on anothermolecule to form a covalent chemical bond between the alkyne modifiedgroup and the alkyne reactive group. Examples of alkyne-reactive groupsinclude azides. “Alkyne-reactive” can also refer to a molecule thatcontains a chemical moiety that selectively reacts with an alkyne group.

The term “antibody” as used herein refers to an immunoglobulin moleculeor immunologically active portion thereof, i.e., an antigen-bindingportion. Examples of immunologically active portions of immunoglobulinmolecules include immunoglobulin molecules or fragments thereof thatcomprise the F(ab) region and a sufficient portion of the Fc region tocomprise the oligosaccharide linkage site, for example, theasparagine-GlcNAc linkage site. An antibody sometimes is a polyclonal,monoclonal, recombinant (e.g., a chimeric or humanized), fully human,non-human (e.g., murine), or a single chain antibody. An antibody mayhave effector function and can fix complement, and is sometimes coupledto a toxin or imaging agent. An antibody is, for example, an IgG.

The term “antibody fragments” as used herein refers to fragments ofantibodies that retain the principal selective binding characteristicsof the whole antibody. Particular fragments are well-known in the art,for example, Fab, Fab′, and F(ab′)₂, which are obtained by digestionwith various proteases, pepsin or papain, and which lack the Fc fragmentof an intact antibody or the so-called “half-molecule” fragmentsobtained by reductive cleavage of the disulfide bonds connecting theheavy chain components in the intact antibody. Such fragments alsoinclude isolated fragments consisting of the light-chain-variableregion, “Fv” fragments consisting of the variable regions of the heavyand light chains, and recombinant single chain polypeptide molecules inwhich light and heavy variable regions are connected by a peptidelinker. Other examples of binding fragments include (i) the Fd fragment,consisting of the VH and CH1 domains; (ii) the dAb fragment (Ward, etal., Nature 341, 544 (1989)), which consists of a VH domain; (iii)isolated CDR regions; and (iv) single-chain Fv molecules (scFv)described above. In addition, arbitrary fragments can be made usingrecombinant technology that retains antigen-recognition characteristics.

The term “antigen” as used herein refers to a molecule that induces, oris capable of inducing, the formation of an antibody or to which anantibody binds selectively, including but not limited to a biologicalmaterial. Antigen also refers to “immunogen”. The target-bindingantibodies selectively bind an antigen, as such the term can be usedherein interchangeably with the term “target”.

The term “anti-region antibody” as used herein refers to an antibodythat was produced by immunizing an animal with a select region that is afragment of a foreign antibody wherein only the fragment is used as theimmunogen. Regions of antibodies include the Fc region, hinge region,Fab region, etc. Anti-region antibodies include monoclonal andpolyclonal antibodies. The term “anti-region fragment” as used hereinrefers to a monovalent fragment that was generated from an anti-regionantibody of the present invention by enzymatic cleavage.

The term “aqueous solution” as used herein refers to a solution that ispredominantly water and retains the solution characteristics of water.Where the aqueous solution contains solvents in addition to water, wateris typically the predominant solvent.

The term “azide reactive” as used herein refers to a chemical moietythat selectively reacts with an azido modified group on another moleculeto form a covalent chemical bond between the azido modified group andthe azide reactive group. Examples of azide-reactive groups includealkynes and phosphines (e.g. triaryl phosphine). “Azide-reactive” canalso refer to a molecule that contains a chemical moiety thatselectively reacts with an azido group.

The term “buffer” as used herein refers to a system that acts tominimize the change in acidity or basicity of the solution againstaddition or depletion of chemical substances.

The term “carrier molecule” as used herein refers to a biological or anon-biological component that is covalently bonded to compound of thepresent invention. Such components include, but are not limited to, anamino acid, a peptide, a protein, a polysaccharide, a nucleoside, anucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, adrug, a hormone, a lipid, a lipid assembly, a synthetic polymer, apolymeric microparticle, a biological cell, a virus and combinationsthereof.

The term, “chemical handle” as used herein refers to a specificfunctional group, such as an azide, alkyne, activated alkyne, phosphite,phosphine, and the like. The chemical handle is distinct from thereactive group, defined below, in that the chemical handle are moietiesthat are rarely found in naturally-occurring biomolecules and arechemically inert towards biomolecules (e.g, native cellular components),but when reacted with an azide- or alkyne-reactive group the reactioncan take place efficiently under biologically relevant conditions (e.g.,cell culture conditions, such as in the absence of excess heat or harshreactants).

The term “click chemistry,” as used herein, refers to the Huisgencycloaddition or the 1,3-dipolar cycloaddition between an azide and aterminal alkyne to form a 1,2,4-triazole. Such chemical reactions canuse, but are not limited to, simple heteroatomic organic reactants andare reliable, selective, stereospecific, and exothermic.

The term “cycloaddition” as used herein refers to a chemical reaction inwhich two or more n-electron systems (e.g., unsaturated molecules orunsaturated parts of the same molecule) combine to form a cyclic productin which there is a net reduction of the bond multiplicity. In acycloaddition, the 7E electrons are used to form new π bonds. Theproduct of a cycloaddition is called an “adduct” or “cycloadduct”.Different types of cycloadditions are known in the art including, butnot limited to, [3+2] cycloadditions and Diels-Alder reactions. [3+2]cycloadditions, which are also called 1,3-dipolar cycloadditions, occurbetween a 1,3-dipole and a dipolarophile and are typically used for theconstruction of five-membered heterocyclic rings. The terms “|3+2|cycloaddition” also encompasses “copperless” [3+2] cycloadditionsbetween azides and cyclooctynes and difluorocyclooctynes described byBertozzi et al. J. Am. Chem. Soc., 2004, 126:15046-15047.

The term “detectable response” as used herein refers to an occurrenceof, or a change in, a signal that is directly or indirectly detectableeither by observation or by instrumentation. Typically, the detectableresponse is an occurrence of a signal wherein the fluorophore isinherently fluorescent and does not produce a change in signal uponbinding to a metal ion or biological compound. Alternatively, thedetectable response is an optical response resulting in a change in thewavelength distribution patterns or intensity of absorbance orfluorescence or a change in light scatter, fluorescence lifetime,fluorescence polarization, or a combination of the above parameters.Other detectable responses include, for example, chemiluminescence,phosphorescence, radiation from radioisotopes, magnetic attraction, andelectron density.

The term “detectably distinct” as used herein refers to a signal that isdistinguishable or separable by a physical property either byobservation or by instrumentation. For example, a fluorophore is readilydistinguishable either by spectral characteristics or by fluorescenceintensity, lifetime, polarization or photo-bleaching rate from anotherfluorophore in the sample, as well as from additional materials that areoptionally present.

The term “directly detectable” as used herein refers to the presence ofa material or the signal generated from the material is immediatelydetectable by observation, instrumentation, or film without requiringchemical modifications or additional substances.

The term “fluorophore” as used herein refers to a composition that isinherently fluorescent or demonstrates a change in fluorescence uponbinding to a biological compound or metal ion, i.e., fluorogenic.Fluorophores may contain substituents that alter the solubility,spectral properties or physical properties of the fluorophore. Numerousfluorophores are known to those skilled in the art and include, but arenot limited to coumarin, cyanine, benzofuran, a quinoline, aquinazolinone, an indole, a benzazole, a borapolyazaindacene andxanthenes including fluoroscein, rhodamine and rhodol as well as otherfluorophores described in RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOKOF FLUORESCENT PROBES AND RESEARCH CHEMICALS (9^(th) edition, CD-ROM,September 2002).

The term “glycomodified” refers to a particle such as a protein orantibody that is altered to change the number or configuration of sugarmolecules attached thereto. A preferred glycomodified antibody is anon-human derived therapeutic antibody that becomes humanized based onthe alteration of its sugar appendages.

The term “glycoprotein” as used herein refers to a protein that has beennaturally glycosolated and those that have been enzymatically modified,in vivo or in vitro, to comprise a sugar group.

The term “humanized” as used herein refers to modification of a protein,such as an antibody, such that it is less immunogenic or invokes areduced immune response in a human as compared to the non-humanizedcounterpart. Preferably, non-human proteins and antibodies are humanizedby modification of sugar groups to be more similar or identical to thoseexpressed in humans.

The term “kit” as used herein refers to a packaged set of relatedcomponents, typically one or more compounds or compositions.

The term “label,” as used herein, refers to a chemical moiety or proteinthat is directly or indirectly detectable (e.g. due to its spectralproperties, conformation or activity) when attached to a target orcompound and used in the present methods, including reporter molecules,solid supports and carrier molecules. As used herein, label ascollectively refers to a reporter molecule, solid support or carriermolecule. The label can be directly detectable (fluorophore) orindirectly detectable (hapten or enzyme). Such labels include, but arenot limited to, radiolabels that can be measured with radiation-countingdevices; pigments, dyes or other chromogens that can be visuallyobserved or measured with a spectrophotometer; spin labels that can bemeasured with a spin label analyzer; and fluorescent labels(fluorophores), where the output signal is generated by the excitationof a suitable molecular adduct and that can be visualized by excitationwith light that is absorbed by the dye or can be measured with standardfluorometers or imaging systems, for example. The label can be achemiluminescent substance, where the output signal is generated bychemical modification of the signal compound; a metal-containingsubstance; or an enzyme, where there occurs an enzyme-dependentsecondary generation of signal, such as the formation of a coloredproduct from a colorless substrate. The term label can also refer to a“tag” or hapten that can bind selectively to a conjugated molecule suchthat the conjugated molecule, when added subsequently along with asubstrate, is used to generate a detectable signal. For example, one canuse biotin as a tag and then use an avidin or streptavidin conjugate ofhorseradish peroxidate (HRP) to bind to the tag, and then use acolorimetric substrate (e.g., tetramethylbenzidine (TMB)) or afluorogenic substrate such as Amplex Red reagent (Molecular Probes,Inc.) to detect the presence of HRP. Numerous labels are know by thoseof skill in the art and include, but are not limited to, particles,fluorophores, haptens, enzymes and their colorimetric, fluorogenic andchemiluminescent substrates and other labels that are described inRICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES ANDRESEARCH PRODUCTS (10^(th) edition, CD-ROM, September 2005), supra.

The term “linker” or “L”, as used herein, refers to a single covalentbond or a series of stable covalent bonds incorporating 1-30 nonhydrogenatoms selected from the group consisting of C, N, O, S and P. Exemplarylinking members include a moiety that includes —C(O)NH—, —C(O)O—, —NH—,—S—, —O—, and the like. A “cleavable linker” is a linker that has one ormore cleavable groups that may be broken by the result of a reaction orcondition. The term “cleavable group” refers to a moiety that allows forrelease of a portion, e.g., a reporter molecule, carrier molecule orsolid support, of a conjugate from the remainder of the conjugate bycleaving a bond linking the released moiety to the remainder of theconjugate. Such cleavage is either chemical in nature, or enzymaticallymediated. Exemplary enzymatically cleavable groups include natural aminoacids or peptide sequences that end with a natural amino acid.

In addition to enzymatically cleavable groups, it is within the scope ofthe present invention to include one or more sites that are cleaved bythe action of an agent other than an enzyme. Exemplary non-enzymaticcleavage agents include, but are not limited to, acids, bases, light(e.g., nitrobenzyl derivatives, phenacyl groups, benzoin esters), andheat. Many cleaveable groups are known in the art. See, for example,Jung et al., Biochem. Biophys. Acta, 761: 152-162 (1983); Joshi et al.,J. Biol. Chem., 265: 14518-14525 (1990); Zarling et al., J. Immunol.,124: 913-920 (1980); Bouizar et al., Eur. J. Biochem., 155: 141-147(1986); Park et al., J. Biol. Chem., 261: 205-210 (1986); Browning etal., J. Immunol., 143: 1859-1867 (1989). Moreover a broad range ofcleavable, bifunctional (both homo- and hetero-bifunctional) spacer armsare commercially available.

An exemplary cleavable group, an ester, is cleavable group that may becleaved by a reagent, e.g. sodium hydroxide, resulting in acarboxylate-containing fragment and a hydroxyl-containing product.

The terms “protein” and “polypeptide” are used herein in a generic senseto include polymers of amino acid residues of any length. The term“peptide” is used herein to refer to polypeptides having less than 100amino acid residues, typically less than 10 amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues are an artificial chemical analogue of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers.

The term “purified” as used herein refers to a preparation of aglycoprotein that is essentially free from contaminating proteins thatnormally would be present in association with the glycoprotein, e.g., ina cellular mixture or milieu in which the protein or complex is foundendogenously such as serum proteins or cellular lysate.

The term “reactive group” as used herein refers to a group that iscapable of reacting with another chemical group to form a covalent bond,i.e. is covalently reactive under suitable reaction conditions, andgenerally represents a point of attachment for another substance. Asused herein, reactive groups refer to chemical moieties generally foundin biological systems and that react under normal biological conditions,these are herein distinguished from the azido and activated alkynemoieties of the present invention. The reactive group is a moiety, suchas carboxylic acid or succinimidyl ester, that is capable of chemicallyreacting with a functional group on a different compound to form acovalent linkage. Reactive groups generally include nucleophiles,electrophiles and photoactivatable groups.

The term “reporter molecule” refers to any moiety capable of beingattached to a modified post translationally modified protein of thepresent invention, and detected either directly or indirectly. Reportermolecules include, without limitation, a chromophore, a fluorophore, afluorescent protein, a phosphorescent dye, a tandem dye, a particle, ahapten, an enzyme and a radioisotope. Preferred reporter moleculesinclude fluorophores, fluorescent proteins, haptens, and enzymes.

The term “sample” as used herein refers to any material that may containan analyte of interest or a modified post translationally modifiedprotein of the present invention . The analyte may include a reactivegroup, e.g., a group through which a compound of the invention can beconjugated to the analyte. The sample may also include diluents,buffers, detergents, and contaminating species, debris and the like thatare found mixed with the target. Illustrative examples include urine,sera, blood plasma, total blood, saliva, tear fluid, cerebrospinalfluid, secretory fluids from nipples and the like. Also included aresolid, gel or sol substances such as mucus, body tissues, cells and thelike suspended or dissolved in liquid materials such as buffers,extractants, solvents and the like. Typically, the sample is a livecell, a biological fluid that comprises endogenous host cell proteins,nucleic acid polymers, nucleotides, oligonucleotides, peptides andbuffer solutions. The sample may be in an aqueous solution, a viablecell culture or immobilized on a solid or semi solid surface such as apolyacrylamide gel, membrane blot or on a microarray.

The term “solid support,” as used herein, refers to a material that issubstantially insoluble in a selected solvent system, or which can bereadily separated (e.g., by precipitation) from a selected solventsystem in which it is soluble. The term solid support includessemi-solid supports. Solid supports useful in practicing the presentinvention can include groups that are activated or capable of activationto allow selected species to be bound to the solid support. Solidsupports may be present in a variety of forms, including a chip, waferor well, onto which an individual, or more than one compound, of theinvention is bound such as a polymeric bead or particle.

The term “Staudinger ligation” as used herein refers to a chemicalreaction developed by Saxon and Bertozzi (E. Saxon and C. Bertozzi,Science, 2000, 287: 2007-2010) that is a modification of the classicalStaudinger reaction. The classical Staudinger reaction is a chemicalreaction in which the combination of an azide with a phosphine orphosphite produces an aza-ylide intermediate, which upon hydrolysisyields a phosphine oxide and an amine A Staudinger reaction is a mildmethod of reducing an azide to an amine; and triphenylphosphine iscommonly used as the reducing agent. In a Staudinger ligation, anelectrophilic trap (usually a methyl ester) is appropriately placed on atriarylphosphine (usually in ortho to the phosphorus atom) and reactedwith the azide, to yield an aza-ylide intermediate, which rearranges inaqueous media to produce a compound with amide group and a phosphineoxide function. The Staudinger ligation is so named because it ligates(attaches/covalently links) the two starting molecules together, whereasin the classical Staudinger reaction, the two products are notcovalently linked after hydrolysis.

The terms “structural integrity of the [biomolecule] is not reduced” or“preservation of the structural integrity of the [biomolecule]”, as usedherein, means that either: 1) when analyzed by gel electrophoresis anddetection (such as staining), a band or spot arising from the labeledbiomolecule is not reduced in intensity by more than 20%, and preferablynot reduced by more than 10%, with respect to the corresponding band orspot arising from the same amount of the electrophoresed unlabeledbiomolecule, arising from the labeled biomolecule analyzed; or 2) whenanalyzed by gel electrophoresis, a band or spot arising from the labeledbiomolecule is not observed to be significantly less sharp than thecorresponding band or spot arising from the same amount of theelectrophoresed unlabeled biomolecule, where “significantly less sharp”(synonymous with “significantly more diffuse”) means the detectable bandor spot takes up at least 5% more, preferably 10% more, more preferably20% more area on the gel than the corresponding unlabeled biomolecule.

Other reproducible tests for structural integrity of labeledbiomolecules include, without limitation detection of released aminoacids or peptides, or mass spectrometry.

By an antibody being “synthesized” in a cell is meant that the antibodyis either naturally produced in, and isolated from, said cell, or thatthe antibody is synthesized using recombinant methods in said cell andis then isolated.

The term “about” as used herein refers to a value sometimes within 10%of the underlying parameter (i.e., plus or minus 10%), a value sometimeswithin 5% of the underlying parameter (i.e., plus or minus 5%), a valuesometimes within 2.5% of the underlying parameter (i.e., plus or minus2.5%), or a value sometimes within 1% of the underlying parameter (i.e.,plus or minus 1%), and sometimes refers to the parameter with novariation. Thus, a distance of “about 20 nucleotides in length” includesa distance of 19 or 21 nucleotides in length (i.e., within a 5%variation) or a distance of 20 nucleotides in length (i.e., novariation) in some embodiments. As used herein, the article “a” or “an”can refer to one or more of the elements it precedes (e.g., a proteinmicroarray “a” protein may comprise one protein sequence or multipleproteins). The term “or” is not meant to be exclusive to one or theterms it designates. For example, as it is used in a phrase of thestructure “A or B” may denote A alone, B alone, or both A and B

In general, for ease of understanding the present invention, themetabolic and enzymatic labeling of biomolecules with azide moieties,alkyne moieties or phosphine, and the chemical labeling of such moietieswith azide reactive moieties, alkyne reactive moieties or phosphinereactive moieties will first be described in detail. This will befollowed by some embodiments in which such labeled biomolecules can bedetected, isolated and/or analyzed. Exemplified methods are thendisclosed.

METHODS OF THE INVENTION Preparation of Antibodies and AntibodyProducing Cells

Antibodies for use in cleavage and substitution of oligosaccharides ofthe present invention may be produced using any means known to those ofordinary skill in the art. General information regarding procedures forantibody production and labeling may be found, for example, inAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chap. 14(1988). Cell lines expressing antibodies for in vivo metabolic labelingmay also be produced using any means known to those of ordinary skill inthe art. For therapeutic purposes, chimeric, humanized, and completelyhuman antibodies are useful for applications that include repeatedadministration to subjects. Chimeric and humanized monoclonalantibodies, comprising both human and non-human portions, can be madeusing standard recombinant DNA techniques. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et alInternational Application No. PCT/US86/02269; Akira, et al EuropeanPatent Application 184, 187; Taniguchi, M., European Patent Application171,496; Morrison et al European Patent Application 173,494; Neubergeret al PCT International Publication No. WO 86/01533; Cabilly et al U.S.Pat. No. 4,816,567; Cabilly et al European Patent Application 125,023;Better et al., Science 240: 1041-1043 (1988); Liu et al., Proc. Natl.Acad. Sci. USA 84: 3439-3443 (1987); Liu et al., J. Immunol 139:3521-3526 (1987); Sun et al., Proc. Natl. Acad. Sci. USA 84: 214-218(1987); Nishimura et al., Canc. Res. 47: 999-1005 (1987); Wood et al.,Nature 314: 446-449 (1985); and Shaw et al., J. Natl. Cancer Inst. 80:1553-1559 (1988); Morrison, S. L., Science 229: 1202-1207 (1985); Oi etal., BioTechniques 4: 214 (1986); Winter U.S. Pat. No. 5,225,539; Joneset al., Nature 321: 552-525 (1986); Verhoeyan et al., Science 239: 1534;and Beidler et al., J. Immunol. 141: 4053-4060 (1988).

Transgenic mice that are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but that can express humanheavy and light chain genes, may be used to produce human antibodies foruse in the present invention. See, for example, Lonberg and Huszar, Int.Rev. Immunol. 13: 65-93 (1995); and U.S. Pat. Nos. 5,625,126; 5,633,425;5,569,825; 5,661,016; and 5,545,806. In addition, companies such asInvitrogen (Carlsbad, Calif.), Abgenix, Inc. (Fremont, Calif.), andMedarex, Inc. (Princeton, N.J.), can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above. Human antibodies that recognize a selectedepitope also can be generated using a technique referred to as “guidedselection.” In this approach a selected non-human monoclonal antibody(e.g., a murine antibody) is used to guide the selection of a completelyhuman antibody recognizing the same epitope. This technology isdescribed for example by Jespers et al., Bio/Technology 12: 899-903(1994).

Glycoproteins:

The glycoprotein constituent of this invention may be any glycoprotein,including for example, hormones, enzymes, antibodies, viral receptors,viral surface glycoproteins, parasite glycoproteins, parasite receptors,T-cell receptors, MHC molecules, immune modifiers, tumor antigens,mucins, inhibitors, growth factors, trophic factors, lymphokines,cytokines, toxoids, nerve growth hormones, blood clotting factors,adhesion molecules, multidrug resistance proteins, adenylate cyclases,bone morphogenic proteins and lectins.

Also included among the glycoproteins are the hormones and cytokines.Examples of hormones include follicle stimulating hormone, humanchorionic gonadotropin, luteinizing hormone, thyrotrophin and ovine,bovine, porcine, murine and rat alleles of these hormones. Examples ofcytokine glycoproteins include .alpha.-interferon, lymphotoxin, andinterleukin-2. Also included are glycoprotein tumor-associated antigens,for example, carcinoembryonic antigen (CEA), human mucins, her-2/neu,and prostate-specific antigen (PSA) [R. A. Henderson and 0. J. Finn,Advances in Immunology, 62, pp. 217-56 (1996)].

Alternatively, the glycoprotein constituent may be selected frompersonal care glycoproteins, including cosmetic glycoproteins,veterinary glycoproteins, food glycoproteins, feed glycoproteins,diagnostic glycoproteins, glycoproteins used in chemical reactions,glycoproteins used in industrial methods, cleaning agent glycoproteins,including detergent glycoproteins, and decontamination glycoproteins.

Included among such glycoproteins are enzymes, such as, for example,hydrolases, transferases, isomerases, lyases, ligases, transferases andoxidoreductases. Examples of hydrolases include lipase, cholinesterase,alkaline phosphatase, .beta.-amylase deoxyribonuclease, glucoamylase Aand B, .alpha.-galactosidase I and II, .beta.-fructofuranosidase,.beta.-glucouronidaDse, N-acetyl-.beta.-glucosaminidase, hyaluronidase,oxytocinase, kallikrein, bromelain, enterokinase, proteinase a, b, andc, pepsinogen and pepsin. Examples of oxidoreductases include glucoseoxidase, peroxidase and chloroperoxidase. Examples of transferasesinclude .gamma.-glutamyltranspeptidase and ribonuclease.

Additional glycoproteins contemplated for use in the present inventioninclude cross-linked glycoproteins, such as those described in U.S. Pat.No. 6,359,118, the contents of which are incorporated by reference.

In Vitro Modification of IgG Oligosaccharides

Oligosaccharides are attached to asparagine residues on immunoglobulingamma (IgG) antibodies by an N-acetylglucosamine disaccharaide linkage(GlcNac-GlcNac). In general, each IgG has two sites near the neck of theIgG where oligosaccharides are attached by GlcNac-GlcNac linkages.Treatment with an endoglycosidase, such as, for example, EndoglycosidaseH (Endo H) cleaves between the two GlcNac sugars. The cleaved end ofeach sugar is referred to as the “reducing end.” By cleaving theoligosaccharide from a first IgG, and collecting the cleavedoligosaccharide, an oligosaccharide having a reducing-end GlcNAc isobtained. A second IgG is then treated with an endoglycosidase, such as,for example, EndoH. This second IgG, which has a GlcNAc having areducing end is then collected. The reducing end of the GlcNAc residueattached to the first oligosaccharide, and the reducing end of theGlcNAc residue oligosaccharide attached to the second IgG may then betreated so that the first oligosaccharide may be attached to the secondIgG.

One method of attaching the first oligosaccharide is, for example, bychemically conjugating the first oligosaccharide to an alkyne. Forexample, the reducing end may be converted to an amine by treatment withammonium bicarbonate, then the alkyne may be attached via a succinimidylester. An azide-modified sugar, such as UDP-GalNAz is then attached tothe reducing end of the GlcNAc residue on the second IgG. This transfermay occur, for example, using a mutant galactosyl transferase. Theazide-labeled IgG is then mixed with the alkyne-labeled oligosaccharide.The alkyne and azide moieties undergo a cycloaddition reaction, whichligates the oligosaccharide from the first IgG to the oligosaccharide onthe second IgG.

Removal of Oligosaccharides from IgG

Oligosaccharides are attached to IgG at asparagine residues on the FcPortion of the antibody (FIG. 1). At the amino acid, there are twoGlcNAc sugars attached to each other by a beta (1-4) linkage. The enzymeEndo-H cleaves this linkage, so that one GlcNAc residue remains attachedto the asparagine on the IgG, while the other GlcNAc remains attached tothe rest of the oligosaccharide. The GlcNAc attached to theoligosaccharide contains a reactive reducing-end, which can beselectively modified without altering the other sugar residues.

Attaching an Azide to the IgG GlcNAc

The enzyme galactosyl transferase normally transfers a galactose fromUDP-galactose to a terminal GlcNAc residue. Khidekel et al (J. Am. Chem.Soc. 2003, 125:16162-16163; Hsieh-Wilson, L., et al., U.S. PatentPublication No. 20050130235, published Jun. 16, 2005, U.S. Ser. No.10/990,767) used a mutant enzyme, a Y289L mutant, to transfer anacetone-containing galactose substrate to a GlcNAc residue. Anazide-containing galactose substrate (UDP-GalNAz) may be synthesized fortransfer to the GlcNAc site by the mutant galactosyl transferase.

Attaching an Alkyne to the Reducing-End of the Cleaved Oligosaccharide

The reducing-end of a sugar is a reactive site that can be selectivelymodified. Some saccharides do not containing reducing ends (such assucrose), rendering them more stable than saccharides with exposedreducing-ends. When the enzyme Endo-H cleaves the GlcNAc-GlcNAc linkage,the reducing end of the oligosaccharide GlcNAc is exposed.

Using the method of Tamura et al. (Analytical Biochem. 1994,216:335-344), the reducing ends of N-linked oligosaccharides may beconverted into amines by reaction with ammonium bicarbonate, resultingin an oligosaccharide-glycosylamine. This reaction may be conductedusing methods known to those of skill in the art, and may, for example,be conducted using a 10-ml glass screw top vial, with about 10micromoles of oligosaccharide in the presence of about 1 gram ofammonium bicarbonate in about 0.5 ml water, and heating the mixture atabout 50° C. for about 24 hours. The resultingoligosaccharide-glycosylamines may be reacted with, for example,acylating agents in order to attach fluorophores, or an N-glycyl linkerwhich itself may be capable of accepting other biological or biophysicalprobes.

For example, a tyrosine may be attached to the amine with aBoc-protected tyrosine succinimidl ester. The Boc protection helps toavoid polymerization of a tyrosine succinimidyl ester. Analkyne-succinimidyl ester is then obtained and may be used in aconjugation scheme. Where a tyrosine residue is attached, theoligosaccharide may be quantified using A280_(nm).

Labeling of Glycoproteins using [3+2] Cycloaddition

Azides and terminal alkynes undergo a cycloaddition reaction in thepresence of a copper catalyst. This cycloaddition reaction may beconducted using the methods of, for example, Sharpless et al. (U.S.Patent Application Publication No. 20050222427, published Oct. 6, 2005,PCT /US03/17311; Lewis W G, et al., Angewandte Chemie-Int'l Ed. 41 (6):1053; method reviewed in Kolb, H. C., et al., Angew. Chem. Inst. Ed.2001, 40:2004-2021), which developed reagents that react with each otherin high yield and with few side reactions in a heteroatom linkage (asopposed to carbon-carbon bonds) in order to create libraries of chemicalcompounds. The reaction is conducted in the presence of a metal catalystand a reducing agent. For example, Cu(II) may be included in thereaction, in the presence of a reducing agent such as, but not limitedto, ascorbate, metallic copper, quinone, hydroquinone, vitamin K₁,glutathione, cysteine, Fe²⁺, Co²⁺, and an applied electric potential.Further preferred reducing agents include metals selected from the groupconsisting of Al, Be, Co, Cr, Fe, Mg, Mn, Ni, and Zn. Other metals thatmay catalyze this type of cycloaddition reaction include, for example,Au, Ag, Hg, Cd, Zr, Ru, Fe, Co, Pt, Pd, Ni, Rh, and W. Those of ordinaryskill in the art would be able to determine the appropriate metal to usefor the intended ligand.

In Vitro Modification of IgG Using EndoM or Endo A

EndoM (or Endo A) are endoglycosidases with glycosyltransferaseactivities. The enzymes cleaves the disaccharide GlcNAc-GlcNAc betweenthe two residues, as does EndoH. EndoM has an additional enzymaticactivity in that once an oligosaccharide having the GlcNAc residue iscleaved, the enzyme will attach the reducing end of the GlcNAc residueto an OH group. Thus, in other methods of the invention, is provided anin vitro method of labeling a cleaved oligosaccharide with an alkyne. Inone example, an IgG comprising an oligosaccharide having a GlcNAc-GlcNAclinkage is incubated in the presence of EndoM as well as an OH-alkyne.

One aspect of the invention provides a method of producing aglycomodified antibody comprising at least one monosaccharide oroligosaccharide, the method comprising:

-   -   contacting an antibody with Endo-M or Endo-A and a donor        comprising the monosaccharide or oligosaccharide to form a        contacted solution, wherein the antibody comprises an acceptor;    -   incubating the contacted solution for a sufficient amount of        time for a covalent bond to form between the acceptor and the        monosaccharide or oligosaccharide; and    -   obtaining the glycomodified antibody.

In a more particular embodiment, the antibody is derived from anon-human source. More particular still, the antibody is derived frommurine cells, plant cells, goat cells, rabbit cells, yeast cells, or CHOcells. In another embodiment, the glycomodified antibody is humanized.

In another embodiment, the acceptor is N-acetylglucosamine (GlcNAc),wherein the GlcNAc. In another embodiment, the antibody is contactedwith Endo-M. In another embodiment, the antibody is contacted withEndo-A. In another embodiment, the donor comprises: Asn-GlcNAc-X,wherein X is the oligosaccharide or monosaccharide. More particularly, Xis -GlcNAc-(oligosaccharide or monosaccharide).

In another embodiment, the monosaccharide or oligosaccharide comprise analkynyl or azido functionality. In another embodiment, themonosaccharide or oligosaccharide comprise a chemical handle.

In another embodiment, the monosaccharide or oligosaccharide comprise areporter molecule. More particularly, the reporter molecule is afluorescent dye, an enzyme, a radiolabel, a metal chelator, or adetectable substrate.

In another embodiment, the monosaccharide or oligosaccharide comprise acarrier molecule, a solid support, a receptor, a ligand, a peptide, aprotein, a nucleic acid polymer, a polysaccharide, an antigen, or ahapten.

In another embodiment, the obtaining step comprises purifying theglycomodified antibody. More particularly, subsequent to purifying, theglycomodified antibody is combined with an excipient, diluent orpharmaceutically acceptable salt. More particular still, theglycomodified antibody combined with an excipient, diluent orpharmaceutically acceptable salt, is administered to a patient in needthereof.

In another embodiment, the glycomodified antibody is a therapeuticantibody. More particularly, the therapeutic antibody is selected fromthe group consisting of trastuzumab, cetuximab, bevacizumab,alemtuzumab, gemtuzumab, ibritumomab, rituximab, and panitumumab.

In another embodiment, the monosaccharide or oligosaccharide affects theserum half life of the glycomodified antibody, targets the glycomodifiedantibody to a particular cell or tissue, or affects the stability of theglycomodified antibody.

In another embodiment, the contacting step is performed in an aqueoussolution. In another embodiment, the contacting step is performed in anorganic solution. In another embodiment, the contacting step isperformed in a protic solution. In another embodiment, the contactingstep is performed in a polar solution. In another embodiment, thecontacting step is performed in a solvent that is not a ketone or furan.

In another embodiment, the glycomodified antibody comprises at least oneoligosaccharide.

In another embodiment, the antibody is a monoclonal antibody. In anotherembodiment, the antibody is a polyconal antibody.

Another aspect of the invention provides a method of covalently bondinga monosaccharide or oligosaccharide to an antibody, the methodcomprising:

-   -   contacting the antibody with Endo-M or Endo-A and a donor        comprising the monosaccharide or oligosaccharide to form a        contacted solution, wherein the antibody comprises an acceptor;        and    -   incubating the contacted solution for a sufficient amount of        time for a covalent bond to form between the acceptor and the        monosaccharide or oligosaccharide.

Another aspect of the invention provides a method of humanizing atherapeutic antibody, the method comprising:

-   -   contacting the therapeutic antibody with Endo-M or Endo-A and a        donor comprising a monosaccharide or oligosaccharide to form a        contacted solution, wherein the antibody comprises an acceptor;        and    -   incubating the contacted solution for a sufficient amount of        time for a covalent bond to form between the acceptor and the        monosaccharide or oligosaccharide, thereby forming a humanized        therapeutic antibody.

In another embodiment, the therapeutic antibody is a monoclonalantibody.

In another embodiment, the therapeutic antibody is selected from thegroup consisting of trastuzumab, cetuximab, bevacizumab, alemtuzumab,gemtuzumab, ibritumomab, rituximab, and panitumumab.

Another aspect of the invention provides a kit for forming a detectableglycomodified antibody comprising:

-   -   (a) Endo-M or Endo-A;    -   (b) a donor comprising an oligosaccharide or monosaccharide; and    -   (c) instructions for forming the detectable glycomodified        antibody.

In another embodiment, the oligosaccharide or monosaccharide comprises areporter group, an azide group, or an alkynyl group.

In another embodiment, the reporter molecule is a fluorescent dye,enzyme, radiolabel, a metal chelator, or a detectable substrate.

Another aspect of the invention provides a composition comprising:

-   -   (a) an antibody comprising an acceptor;    -   (b) Endo-M or Endo-A;    -   (c) a donor comprising an oligosaccharide or monosaccharide; and    -   (d) a solvent.

Metabolic Labeling of Antibodies in Antibody Producing Cells

In vivo metabolic labeling methods may be used to label antibodies inantibody-producing cells such as, for example, hybridoma cells, and anyother cell that produces antibodies or recombinant antibodies.Antibody-producing cells may be fed unnatural sugar substrates, forexample unnatural sugar substrates that contain a reactivechemical/affinity handle. The chemical handles may be, for example, butnot limited to, azides, triarylphosphines, or alkyne residues that may,for example, participate in “click” chemistry type reactions. In certainembodiments, the unnatural sugars comprise modifications that are smallenough to be incorporated into the cells. The cellular metabolicmachinery incorporates the substrates into N- or O-linked glycansattached to the antibodies. Once the in vivo labeled antibodies arereleased and isolated from the cells, the labeled antibodies may bedirectly labeled using a detection/affinity/immobilization compound thatis reactive with the chemical handles. These compounds may include, forexample, but are not limited to, fluorophores, solid support resins,microarray slides, or affinity tags.

Metabolic Labeling of Antibodies

Antibody-producing cells are incubated in the presence of unnaturalsugars, for example, using the methods of Bertozzi et al., U.S. Pat. No.6,936,701, which provides examples of incubating cells in the presenceof unnatural sugars to modify cell-surface glycoproteins. The cells maybe incubated with, for example, about 20 mM of the unnatural sugar for,for example, about 72 hours.

Synthesis of Unnatural Sugar Substrates

Unnatural sugar substrates may be synthesized that incorporate reactivechemical handles that may be used for click chemistry. The azide/alkynecycloaddition reaction can be used to introduce affinity probes(biotin), dyes, polymers (e.g., poly(ethylene glycol) or polydextran) orother monosaccharides (e.g., glucose, galactose, fucose, O-GlcNAc,mannose-derived saccharides bearing the appropriate chemical handle). Incertain embodiments, these handles include, for example, azide,triarylphosphine, or alkyne residues. The chemical handle also can be anazido group capable of reacting in a Staudinger reaction (see, forexample, Saxon, E., et al., J. Am. Chem. Soc., 2002,124(50):14893-14902). The Staudinger reaction involves reaction betweentrivalent phosphorous compounds and organic azides (Staudinger et al.,Helv. Chim Acta 1919, 2:635), has been used for a multitude ofapplications. (Gololobov et al. Tetrahedron 1980, 37, 437); (Gololobovet al. Tetrahedron 1992, 48, 1353). The phosphine can have a neighboringacyl group such as an ester, thioester or N-acyl imidazole (i.e. aphosphinoester, phosphinothioester, phosphinoimidazole) to trap theaza-ylide intermediate and form a stable amide bond upon hydrolysis. Thephosphine can also be typically a di- or triarylphosphine to stabilizethe phosphine.

Unnatural sugar substrates that may be incorporated in vivo according tothe present invention include, for example, but are not limited to,GalNAz, ManNaz, and GlcNAz. Unnatural sugar substrates include, forexample, sugar substrates that comprise small sugar groups that thecellular machinery would be more likely to incorporate, and notrecognize as being foreign.

Labeling Cleaved Oligosaccharides and In Vivo Labeled IgGs

Various labels, or tags, (reporter molecule, solid support and carriermolecule) may be linked or conjugated to the cleaved oligosaccharide forthe in vitro methods of the present invention, which would then beattached to the cleaved IgGs of the methods of the present invention.These labels or tags may also be used to label or tag the in vivo-sugarmodified IgGs isolated from the cells after metabolic labeling; thelabels or tags may be attached at the chemical/affinity handles on theunnatural sugars incorporated during metabolic labeling.

The labels or tags may be, for example, a therapeutic moiety such as acytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxinor cytotoxic agent includes any agent that is detrimental to cells.Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide,emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents include,but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thiotepachlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU),cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Antibody conjugates can be used for modifying a given biologicalresponse. For example, the drug moiety may be DNA or a protein orpolypeptide possessing a desired biological activity. Such proteins mayinclude, for example, a toxin such as abrin, ricin A, pseudomonasexotoxin, or diphtheria toxin; a polypeptide such as tumor necrosisfactor, gamma.-interferon, .alpha.-interferon, nerve growth factor,platelet derived growth factor, tissue plasminogen activator; or,biological response modifiers such as, for example, lymphokines,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocytecolony stimulating factor (“G-CSF”), or other growth factors. Also, anantibody can be conjugated to a second antibody to form an antibodyheteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, forexample.

The labels or tags may also, for example, be detectable labels used, forexample, for diagnostic or research purposes, or tags used for bindingof the antibody to other biomolecules or reagents (such as, for example,biotin/avidin binding, or binding of the antibody to microarray chips,beads, or plates). Examples of such labels or tags include, but are notlimited to fluorescent dyes, such as, for example, fluorescein (FITC),Oregon Green 488 dye, Marina Blue dye, Pacific Blue dye, and Texas Red-Xdye, Alexa Fluor dyes (Invitrogen, Carlsbad, Calif.); compoundscontaining radioisotopes; light-scattering compounds such as, forexample, those containing gold or silver; dyes; light producingcompounds such as, for example, luciferase; haptens, such as, forexample, biotin, desthiobiotin, DSB-X biotin, and dinitrophenol (DNP);enzymes, such as, for example, horseradish peroxidase (HRP), alkalinephosphatase, and [beta]-lactamase; phycobiliproteins, such as, forexample, R-phycoerythrin (R-PE, allophycocyanin (AP); and particles,such as, for example, Qdots, gold, ferrofluids, dextrans andmicrospheres.

Reporter Molecules:

The reporter molecules of the present invention include any directly orindirectly detectable reporter molecule known by one skilled in the artthat can be attached to a modified glycoprotein of the presentinvention. Reporter molecules include, without limitation, achromophore, a fluorophore, a fluorescent protein, a phosphorescent dye,a tandem dye, a particle, a hapten, an enzyme and a radioisotope.Preferred reporter molecules include fluorophores, fluorescent proteins,haptens, and enzymes.

A fluorophore of the present invention is any chemical moiety thatexhibits an absorption maximum beyond 280 nm, and when covalentlyattached to a labeling reagent retains its spectral properties.Fluorophores of the present invention include, without limitation; apyrene (including any of the corresponding derivative compoundsdisclosed in U.S. Pat. No. 5,132,432), an anthracene, a naphthalene, anacridine, a stilbene, an indole or benzindole, an oxazole orbenzoxazole, a thiazole or benzothiazole, a4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a cyanine (including anycorresponding compounds in U.S. Ser. Nos. 09/968,401 and 09/969,853), acarbocyanine (including any corresponding compounds in U.S. Ser. Nos.09/557,275; 09/969,853 and 09/968,401; U.S. Pat. Nos. 4,981,977;5,268,486; 5,569,587; 5,569,766; 5,486,616; 5,627,027; 5,808,044;5,877,310; 6,002,003; 6,004,536; 6,008,373; 6,043,025; 6,127,134;6,130,094; 6,133,445; and publications WO 02/26891, WO 97/40104, WO99/51702, WO 01/21624; EP 1 065 250 A1), a carbostyryl, a porphyrin, asalicylate, an anthranilate, an azulene, a perylene, a pyridine, aquinoline, a borapolyazaindacene (including any corresponding compoundsdisclosed in U.S. Pat. Nos. 4,774,339; 5,187,288; 5,248,782; 5,274,113;and 5,433,896), a xanthene (including any corresponding compoundsdisclosed in U.S. Pat. No. 6,162,931; 6,130,101; 6,229,055; 6,339,392;5,451,343 and U.S. Ser. No. 09/922,333), an oxazine (including anycorresponding compounds disclosed in U.S. Pat. No. 4,714,763) or abenzoxazine, a carbazine (including any corresponding compoundsdisclosed in U.S. Pat. No. 4,810,636), a phenalenone, a coumarin(including an corresponding compounds disclosed U.S. Pat. Nos.5,696,157; 5,459,276; 5,501,980 and 5,830,912), a benzofuran (includingan corresponding compounds disclosed U.S. Pat. Nos. 4,603,209 and4,849,362) and benzphenalenone (including any corresponding compoundsdisclosed in U.S. Pat. No. 4,812,409) and derivatives thereof. As usedherein, oxazines include resorufins (including any correspondingcompounds disclosed in U.S. Pat. No. 5,242,805), aminooxazinones,diaminooxazines, and their benzo-substituted analogs.

When the fluorophore is a xanthene, the fluorophore is optionally afluorescein, a rhodol (including any corresponding compounds disclosedin U.S. Pat. Nos. 5,227,487 and 5,442,045), or a rhodamine (includingany corresponding compounds in U.S. Pat. Nos. 5,798,276; 5,846,737; U.S.Ser. No. 09/129,015). As used herein, fluorescein includes benzo- ordibenzofluoresceins, seminaphthofluoresceins, or naphthofluoresceins.Similarly, as used herein rhodol includes seminaphthorhodafluors(including any corresponding compounds disclosed in U.S. Pat. No.4,945,171). Alternatively, the fluorophore is a xanthene that is boundvia a linkage that is a single covalent bond at the 9-position of thexanthene.

Preferred xanthenes include derivatives of 3H-xanthen-6-ol-3-oneattached at the 9-position, derivatives of 6-amino-3H-xanthen-3-oneattached at the 9-position, or derivatives of 6-amino-3H-xanthen-3-imineattached at the 9-position.

Preferred fluorophores of the invention include xanthene (rhodol,rhodamine, fluorescein and derivatives thereof) coumarin, cyanine,pyrene, oxazine and borapolyazaindacene. Most preferred are sulfonatedxanthenes, fluorinated xanthenes, sulfonated coumarins, fluorinatedcoumarins and sulfonated cyanines. The choice of the fluorophoreattached to the labeling reagent will determine the absorption andfluorescence emission properties of the labeling reagent andimmuno-labeled complex. Physical properties of a fluorophore labelinclude spectral characteristics (absorption, emission and stokesshift), fluorescence intensity, lifetime, polarization andphoto-bleaching rate all of which can be used to distinguish onefluorophore from another.

Typically the fluorophore contains one or more aromatic orheteroaromatic rings, that are optionally substituted one or more timesby a variety of substituents, including without limitation, halogen,nitro, cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl,cycloalkyl, arylalkyl, acyl, aryl or heteroaryl ring system, benzo, orother substituents typically present on fluorophores known in the art.

In one aspect of the invention, the fluorophore has an absorptionmaximum beyond 480 nm. In a particularly useful embodiment, thefluorophore absorbs at or near 488 nm to 514 nm (particularly suitablefor excitation by the output of the argon-ion laser excitation source)or near 546 nm (particularly suitable for excitation by a mercury arclamp).

Many of fluorophores can also function as chromophores and thus thedescribed fluorophores are also preferred chromophores of the presentinvention.

In addition to fluorophores, enzymes also find use as labels for thedetection reagents. Enzymes are desirable labels because amplificationof the detectable signal can be obtained resulting in increased assaysensitivity. The enzyme itself does not produce a detectable responsebut functions to break down a substrate when it is contacted by anappropriate substrate such that the converted substrate produces afluorescent, colorimetric or luminescent signal. Enzymes amplify thedetectable signal because one enzyme on a labeling reagent can result inmultiple substrates being converted to a detectable signal. This isadvantageous where there is a low quantity of target present in thesample or a fluorophore does not exist that will give comparable orstronger signal than the enzyme. However, fluorophores are mostpreferred because they do not require additional assay steps and thusreduce the overall time required to complete an assay. The enzymesubstrate is selected to yield the preferred measurable product, e.g.colorimetric, fluorescent or chemiluminescence. Such substrates areextensively used in the art, many of which are described in theMOLECULAR PROBES HANDBOOK, supra.

A preferred colorimetric or fluorogenic substrate and enzyme combinationuses oxidoreductases such as horseradish peroxidase and a substrate suchas 3,3′-diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC), whichyield a distinguishing color (brown and red, respectively). Othercolorimetric oxidoreductase substrates that yield detectable productsinclude, but are not limited to:2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),o-phenylenediamine (OPD), 3,3′,5,5′-tetramethylbenzidine (TMB),o-dianisidine, 5-aminosalicylic acid, 4-chloro-1-naphthol. Fluorogenicsubstrates include, but are not limited to, homovanillic acid or4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines and reducedbenzothiazines, including Amplex® Red reagent and its variants (U.S.Pat. No. 4,384,042), Amplex UltraRed and its variants in (WO05042504)and reduced dihydroxanthenes, including dihydrofluoresceins (U.S. Pat.No. 6,162,931) and dihydrorhodamines including dihydrorhodamine 123.Peroxidase substrates that are tyramides (U.S. Pat. Nos. 5,196,306;5,583,001 and 5,731,158) represent a unique class of peroxidasesubstrates in that they can be intrinsically detectable before action ofthe enzyme but are “fixed in place” by the action of a peroxidase in theprocess described as tyramide signal amplification (TSA). Thesesubstrates are extensively utilized to label targets in samples that arecells, tissues or arrays for their subsequent detection by microscopy,flow cytometry, optical scanning and fluorometry.

Another preferred colorimetric (and in some cases fluorogenic) substrateand enzyme combination uses a phosphatase enzyme such as an acidphosphatase, an alkaline phosphatase or a recombinant version of such aphosphatase in combination with a colorimetric substrate such as5-bromo-6-chloro-3-indolyl phosphate (BCIP), 6-chloro-3-indolylphosphate, 5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenylphosphate, or o-nitrophenyl phosphate or with a fluorogenic substratesuch as 4-methylumbelliferyl phosphate,6,8-difluoro-7-hydroxy-4-methylcoumarinyl phosphate (DiFMUP, U.S. Pat.No. 5,830,912) fluorescein diphosphate, 3-O-methylfluorescein phosphate,resorufin phosphate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)phosphate (DDAO phosphate), or ELF 97, ELF 39 or related phosphates(U.S. Pat. Nos. 5,316,906 and 5,443,986).

Glycosidases, in particular beta-galactosidase, beta-glucuronidase andbeta-glucosidase, are additional suitable enzymes. Appropriatecolorimetric substrates include, but are not limited to,5-bromo-4-chloro-3-indolylbeta-D-galactopyranoside (X-gal) and similarindolyl galactosides, glucosides, and glucuronides, o-nitrophenylbeta-D-galactopyranoside (ONPG) and p-nitrophenylbeta-D-galactopyranoside. Preferred fluorogenic substrates includeresorufin beta-D-galactopyranoside, fluorescein digalactoside (FDG),fluorescein diglucuronide and their structural variants (U.S. Pat. Nos.5,208,148; 5,242,805; 5,362,628; 5,576,424 and 5,773,236),4-methylumbelliferyl beta-D-galactopyranoside, carboxyumbelliferylbeta-D-galactopyranoside and fluorinated coumarinbeta-D-galactopyranosides (U.S. Pat. No. 5,830,912).

Additional enzymes include, but are not limited to, hydrolases such ascholinesterases and peptidases, oxidases such as glucose oxidase andcytochrome oxidases, and reductases for which suitable substrates areknown.

Enzymes and their appropriate substrates that produce chemiluminescenceare preferred for some assays. These include, but are not limited to,natural and recombinant forms of luciferases and aequorins.Chemiluminescence-producing substrates for phosphatases, glycosidasesand oxidases such as those containing stable dioxetanes, luminol,isoluminol and acridinium esters are additionally useful.

In addition to enzymes, haptens such as biotin are also preferredlabels. Biotin is useful because it can function in an enzyme system tofurther amplify the detectable signal, and it can function as a tag tobe used in affinity chromatography for isolation purposes. For detectionpurposes, an enzyme conjugate that has affinity for biotin is used, suchas avidin-HRP. Subsequently a peroxidase substrate is added to produce adetectable signal.

Haptens also include hormones, naturally occurring and synthetic drugs,pollutants, allergens, affector molecules, growth factors, chemokines,cytokines, lymphokines, amino acids, peptides, chemical intermediates,nucleotides and the like.

Fluorescent proteins also find use as labels for the labeling reagentsof the present invention. Examples of fluorescent proteins include greenfluorescent protein (GFP) and the phycobiliproteins and the derivativesthereof. The fluorescent proteins, especially phycobiliprotein, areparticularly useful for creating tandem dye labeled labeling reagents.These tandem dyes comprise a fluorescent protein and a fluorophore forthe purposes of obtaining a larger stokes shift wherein the emissionspectra is farther shifted from the wavelength of the fluorescentprotein's absorption spectra. This is particularly advantageous fordetecting a low quantity of a target in a sample wherein the emittedfluorescent light is maximally optimized, in other words little to noneof the emitted light is reabsorbed by the fluorescent protein. For thisto work, the fluorescent protein and fluorophore function as an energytransfer pair wherein the fluorescent protein emits at the wavelengththat the fluorophore absorbs at and the fluorophore then emits at awavelength farther from the fluorescent proteins than could have beenobtained with only the fluorescent protein. A particularly usefulcombination is the phycobiliproteins disclosed in U.S. Pat. Nos.4,520,110; 4,859,582; 5,055,556 and the sulforhodamine fluorophoresdisclosed in U.S. Pat. No. 5,798,276, or the sulfonated cyaninefluorophores disclosed in U.S. Ser. Nos. 09/968,401 and 09/969,853; orthe sulfonated xanthene derivatives disclosed in U.S. Pat. No. 6,130,101and those combinations disclosed in U.S. Pat. No. 4,542,104.Alternatively, the fluorophore functions as the energy donor and thefluorescent protein is the energy acceptor.

In an exemplary embodiment is provided glycoproteins covalentlyconjugated to a carrier molecule. This includes, but is not limited to,any azide modified glycoprotein and any carrier molecule disclosedherein.

Provided in one embodiment is a first composition that comprises apresent glycoprotein, a first reporter molecule, and a carrier molecule.Provided in another embodiment is a second glycoprotein that includes afirst composition in combination with a second conjugate. The secondconjugate comprises a carrier molecule or solid support, as disclosedbelow, that is covalently bonded to a second reporter molecule. Thefirst and second reporter molecules have different structures andpreferably have different emission spectra. Even more preferably, thefirst and second reporter molecules are selected so that theirfluorescence emissions essentially do not overlap. In another embodimentthe reporter molecules have different excitation spectra, alternativelythe reporter molecules are excited by the same laser.

Carrier Molecules:

The carrier molecule (or solid support) of the conjugates in the secondcomposition may be the same or a different molecule. The discussionherein pertaining to the identity of various carrier molecules isgenerally applicable to this embodiment of the invention as well asother embodiments.

A variety of carrier molecules are useful in the present invention.Exemplary carrier molecules include antigens, steroids, vitamins, drugs,haptens, metabolites, toxins, environmental pollutants, amino acids,peptides, proteins, nucleic acids, nucleic acid polymers, carbohydrates,lipids, and polymers.

In an exemplary embodiment, the carrier molecule comprises an aminoacid, a peptide, a protein, a polysaccharide, a nucleoside, anucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, adrug, a hormone, a lipid, a lipid assembly, a synthetic polymer, apolymeric microparticle, a biological cell, a virus and combinationsthereof. In another exemplary embodiment, the carrier molecule isselected from a hapten, a nucleotide, an oligonucleotide, a nucleic acidpolymer, a protein, a peptide or a polysaccharide. In a preferredembodiment the carrier molecule is amino acid, a peptide, a protein, apolysaccharide, a nucleoside, a nucleotide, an oligonucleotide, anucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipidassembly, a tyramine, a synthetic polymer, a polymeric microparticle, abiological cell, cellular components, an ion chelating moiety, anenzymatic substrate or a virus. In another preferred embodiment, thecarrier molecule is an antibody or fragment thereof, an antigen, anavidin or streptavidin, a biotin, a dextran, an IgG binding protein, afluorescent protein, agarose, and a non-biological microparticle.

In an exemplary embodiment, the enzymatic substrate is selected from anamino acid, peptide, sugar, alcohol, alkanoic acid, 4-guanidinobenzoicacid, nucleic acid, lipid, sulfate, phosphate, —CH₂OCOalkyl andcombinations thereof. Thus, the enzyme substrates can be cleave byenzymes selected from the group consisting of peptidase, phosphatase,glycosidase, dealkylase, esterase, guanidinobenzotase, sulfatase,lipase, peroxidase, histone deacetylase, endoglycoceramidase,exonuclease, reductase and endonuclease.

In another exemplary embodiment, the carrier molecule is an amino acid(including those that are protected or are substituted by phosphates,carbohydrates, or C₁ to C₂₂ carboxylic acids), or a polymer of aminoacids such as a peptide or protein. In a related embodiment, the carriermolecule contains at least five amino acids, more preferably 5 to 36amino acids. Exemplary peptides include, but are not limited to,neuropeptides, cytokines, toxins, protease substrates, and proteinkinase substrates. Other exemplary peptides may function as organellelocalization peptides, that is, peptides that serve to target theconjugated compound for localization within a particular cellularsubstructure by cellular transport mechanisms. Preferred protein carriermolecules include enzymes, antibodies, lectins, glycoproteins, histones,albumins, lipoproteins, avidin, streptavidin, protein A, protein G,phycobiliproteins and other fluorescent proteins, hormones, toxins andgrowth factors. Typically, the protein carrier molecule is an antibody,an antibody fragment, avidin, streptavidin, a toxin, a lectin, or agrowth factor. Exemplary haptens include biotin, digoxigenin andfluorophores.

In another exemplary embodiment, the carrier molecule comprises anucleic acid base, nucleoside, nucleotide or a nucleic acid polymer,optionally containing an additional linker or spacer for attachment of afluorophore or other ligand, such as an alkynyl linkage (U.S. Pat. No.5,047,519), an aminoallyl linkage (U.S. Pat. No. 4,711,955) or otherlinkage. In another exemplary embodiment, the nucleotide carriermolecule is a nucleoside or a deoxynucleoside or a dideoxynucleoside.

Exemplary nucleic acid polymer carrier molecules are single- ormulti-stranded, natural or synthetic DNA or RNA oligonucleotides, orDNA/RNA hybrids, or incorporating an unusual linker such as morpholinederivatized phosphates (AntiVirals, Inc., Corvallis Oreg.), or peptidenucleic acids such as N-(2-aminoethyl)glycine units, where the nucleicacid contains fewer than 50 nucleotides, more typically fewer than 25nucleotides.

In another exemplary embodiment, the carrier molecule comprises acarbohydrate or polyol that is typically a polysaccharide, such asdextran, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, starch,agarose and cellulose, or is a polymer such as a poly(ethylene glycol).In a related embodiment, the polysaccharide carrier molecule includesdextran, agarose or FICOLL.

In another exemplary embodiment, the carrier molecule comprises a lipid(typically having 6-25 carbons), including glycolipids, phospholipids,and sphingolipids. Alternatively, the carrier molecule comprises a lipidvesicle, such as a liposome, or is a lipoprotein (see below). Somelipophilic substituents are useful for facilitating transport of theconjugated dye into cells or cellular organelles.

Alternatively, the carrier molecule is a cell, cellular systems,cellular fragment, or subcellular particles, including virus particles,bacterial particles, virus components, biological cells (such as animalcells, plant cells, bacteria, or yeast), or cellular components.Examples of cellular components that are useful as carrier moleculesinclude lysosomes, endosomes, cytoplasm, nuclei, histones, mitochondria,Golgi apparatus, endoplasmic reticulum and vacuoles.

In another exemplary embodiment, the carrier molecule non-covalentlyassociates with organic or inorganic materials. Exemplary embodiments ofthe carrier molecule that possess a lipophilic substituent can be usedto target lipid assemblies such as biological membranes or liposomes bynon-covalent incorporation of the dye compound within the membrane,e.g., for use as probes for membrane structure or for incorporation inliposomes, lipoproteins, films, plastics, lipophilic microspheres orsimilar materials.

In an exemplary embodiment, the carrier molecule comprises a specificbinding pair member wherein the present compounds are conjugated to aspecific binding pair member and used to the formation of the boundpair. Alternatively, the presence of the labeled specific binding pairmember indicates the location of the complementary member of thatspecific binding pair; each specific binding pair member having an areaon the surface or in a cavity which specifically binds to, and iscomplementary with, a particular spatial and polar organization of theother. In this instance, the dye compounds of the present inventionfunction as a reporter molecule for the specific binding pair. Exemplarybinding pairs are set forth in Table 2.

TABLE 2 Representative Specific Binding Pairs antigen antibody biotinavidin (or streptavidin or anti-biotin) IgG* protein A or protein G drugdrug receptor folate folate binding protein toxin toxin receptorcarbohydrate lectin or carbohydrate receptor peptide peptide receptorprotein protein receptor enzyme substrate enzyme DNA (RNA) cDNA (cRNA)†hormone hormone receptor ion chelator *IgG is an immunoglobulin †cDNAand cRNA are the complementary strands used for hybridization

In a particular aspect the carrier molecule is an antibody fragment,such as, but not limited to, anti-Fc, an anti-Fc isotype, anti-J chain,anti-kappa light chain, anti-lambda light chain, or a single-chainfragment variable protein; or a non-antibody peptide or protein, suchas, for example but not limited to, soluble Fc receptor, protein G,protein A, protein L, lectins, or a fragment thereof. In one aspect thecarrier molecule is a Fab fragment specific to the Fc portion of thetarget-binding antibody or to an isotype of the Fc portion of thetarget-binding antibody (U.S. Ser. No. 10/118,204). The monovalent Fabfragments are typically produced from either murine monoclonalantibodies or polyclonal antibodies generated in a variety of animals,for example but not limited to, rabbit or goat. These fragments can begenerated from any isotype such as murine IgM, IgG₁, IgG_(2a), IgG_(2b)or IgG₃.

Alternatively, a non-antibody protein or peptide such as protein G, orother suitable proteins, can be used alone or coupled with albumin.Preferred albumins include human and bovine serum albumins or ovalbuminProtein A, G and L are defined to include those proteins known to oneskilled in the art or derivatives thereof that comprise at least onebinding domain for IgG, i.e. proteins that have affinity for IgG. Theseproteins can be modified but do not need to be and are conjugated to areactive label in the same manner as the other carrier molecules of theinvention.

In another aspect the carrier molecule is a whole intact antibody.Antibody is a term of the art denoting the soluble substance or moleculesecreted or produced by an animal in response to an antigen, and whichhas the particular property of combining specifically with the antigenthat induced its formation. Antibodies themselves also serve areantigens or immunogens because they are glycoproteins and therefore areused to generate anti-species antibodies. Antibodies, also known asimmunoglobulins, are classified into five distinct classes—IgG, IgA,IgM, IgD, and IgE. The basic IgG immunoglobulin structure consists oftwo identical light polypeptide chains and two identical heavypolypeptide chains (linked together by disulfide bonds).

When IgG is treated with the enzyme papain a monovalent antigen-bindingfragment can be isolated, referred herein to as a Fab fragment. When IgGis treated with pepsin (another proteolytic enzyme), a larger fragmentis produced, F(ab′)₂. This fragment can be split in half by treatingwith a mild reducing buffer that results in the monovalent Fab′fragment. The Fab′ fragment is slightly larger than the Fab and containsone or more free sulfhydryls from the hinge region (which are not foundin the smaller Fab fragment). The term “antibody fragment” is usedherein to define the Fab′, F(ab′)₂ and Fab portions of the antibody. Itis well known in the art to treat antibody molecules with pepsin andpapain in order to produce antibody fragments (Gorevic et al., Methodsof Enzyol., 116:3 (1985)).

The monovalent Fab fragments of the present invention are produced fromeither murine monoclonal antibodies or polyclonal antibodies generatedin a variety of animals that have been immunized with a foreign antibodyor fragment thereof, U.S. Pat. No. 4,196,265 discloses a method ofproducing monoclonal antibodies. Typically, secondary antibodies arederived from a polyclonal antibody that has been produced in a rabbit orgoat but any animal known to one skilled in the art to producepolyclonal antibodies can be used to generate anti-species antibodies.The term “primary antibody” describes an antibody that binds directly tothe antigen as opposed to a “secondary antibody” that binds to a regionof the primary antibody. Monoclonal antibodies are equal, and in somecases, preferred over polyclonal antibodies provided that theligand-binding antibody is compatible with the monoclonal antibodiesthat are typically produced from murine hybridoma cell lines usingmethods well known to one skilled in the art.

In one aspect the antibodies are generated against only the Fc region ofa foreign antibody. Essentially, the animal is immunized with only theFc region fragment of a foreign antibody, such as murine. The polyclonalantibodies are collected from subsequent bleeds, digested with anenzyme, pepsin or papain, to produce monovalent fragments. The fragmentsare then affinity purified on a column comprising whole immunoglobulinprotein that the animal was immunized against or just the Fc fragments.

In an exemplary embodiment is provided azido glycoproteins covalentlyconjugated to a solid support. This includes, but is not limited to, anyazido glycoprotein disclosed above and any solid support disclosedherein.

Provided in one embodiment is a first composition that comprises apresent compound, a first reporter molecule, and a solid support.Provided in another embodiment is a second composition that includes afirst composition in combination with a second conjugate. The secondconjugate comprises a solid support or carrier molecule, as disclosedabove, that is covalently bonded to a second reporter molecule. Thefirst and second reporter molecules have different structures andpreferably have different emission spectra. Even more preferably, thefirst and second reporter molecules are selected so that theirfluorescence emissions essentially do not overlap. In another embodimentthe reporter molecule have different excitation spectra, alternativelythe reporter molecules are excited by the same laser.

The solid support (or carrier molecule) of the conjugates in the secondcomposition may be the same or a different molecule. The discussionherein pertaining to the identity of various solid supports is generallyapplicable to this embodiment of the invention as well as otherembodiments.

Solid Supports:

A variety of solid supports are useful in the present invention. A solidsupport suitable for use in the present invention is typicallysubstantially insoluble in liquid phases and contains a substituentcapable of reacting with a chemical handle. In a preferred embodiment,the solid supports comprise an alkyne or activated alkyne and react withan azide on a modified sugar, or vice versa. Solid supports of thecurrent invention are not limited to a specific type of support andinclude semi-solid supports. Rather, a large number of supports areavailable and are known to one of ordinary skill in the art. Thus,useful solid supports include solid and semi-solid matrixes, such asaerogels and hydrogels, resins, beads, biochips (including thin filmcoated biochips), microfluidic chip, a silicon chip, multi-well plates(also referred to as microtitre plates or microplates), membranes,conducting and nonconducting metals, glass (including microscope slides)and magnetic supports. More specific examples of useful solid supportsinclude silica gels, polymeric membranes, particles, derivatized plasticfilms, glass beads, cotton, plastic beads, alumina gels, polysaccharidessuch as Sepharose, poly(acrylate), polystyrene, poly(acrylamide),polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin,glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose,polyvinylchloride, polypropylene, polyethylene (including poly(ethyleneglycol)), nylon, latex bead, magnetic bead, paramagnetic bead,superparamagnetic bead, starch and the like.

In some embodiments, the solid support may include a solid supportreactive functional group, including, but not limited to, hydroxyl,carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea,carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide,sulfoxide, etc., for attaching the compounds of the invention. Usefulreactive groups are disclosed above and are equally applicable to thesolid support reactive functional groups herein.

A suitable solid phase support can be selected on the basis of desiredend use and suitability for various synthetic protocols. For example,where amide bond formation is desirable to attach the compounds of theinvention to the solid support, resins generally useful in peptidesynthesis may be employed, such as polystyrene (e.g., PAM-resin obtainedfrom Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE™ resin(obtained from Aminotech, Canada), polyamide resin (obtained fromPeninsula Laboratories), polystyrene resin grafted with polyethyleneglycol (TentaGel™, Rapp Polymere, Tubingen, Germany),polydimethyl-acrylamide resin (available from Milligen/Biosearch,California), or PEGA beads (obtained from Polymer Laboratories).

Kits:

Kits are provided that comprise components for in vivo metaboliclabeling of proteins and antibodies. Such kits may comprise, forexample, unnatural sugars labeled with a chemical/affinity handle, suchas, for example, azides, triarylphosphines, or alkynes. Such kits mayfurther comprise, for example, reagents for labeling or taggingmetabolically labeled antibodies isolated from cells. For example, a kitmay comprise an unnatural oligosaccharide, such as, for example, GalNAz,ManNAz, or GlcNAz. The kit may further comprise a fluorophore label, oran affinity tag, that is reactive with the chemical/affinity handle onthe unnatural oligosaccharide. Those of ordinary skill in the art areaware of the various labels or tags that may be used to label or tag themodified antibodies, for example, but not limited to, those listedherein. For example, the label or tag may be linked to an alkyne, thatis capable of a click chemistry-type reaction once contacted with theazide on the metabolically-labeled antibody.

The kits of the present invention may also comprise one or more of thecomponents in any number of separate containers, packets, tubes, vials,microtiter plates and the like, or the components may be combined invarious combinations in such containers. For the kits of the presentinvention, for example, the unnatural oligosaccharide, comprising thechemical/affinity handle may be provided in a separate container thanthe fluorophore label or affinity tag.

The kits of the present invention may also comprise instructions forperforming one or more methods described herein and/or a description ofone or more compositions or reagents described herein. Instructionsand/or descriptions may be in printed form and may be included in a kitinsert. A kit also may include a written description of an Internetlocation that provides such instructions or descriptions.

Particular Aspects of the Invention:

The methods of the present invention can be used for directly labelingthe terminal saccharide residue on a glycoprotein or antibody, such aswith the Gal-T enzyme described herein. Alternatively, a saccharidegroup is cleaved, such as with Endo-H, and then a saccharide comprisingthe chemical handle is added to the protein or antibody, such as withEndo-M or Endo-A (both of which are capable of cleavage and transfer ofthe sugar). In another embodiment, a saccharide comprising a chemicalhandle is introduced to a glycoprotein or antibody through metaboliclabeling. Reference to “saccharide” indicates monosaccharide oroligosaccharide.

One aspect of the invention provides a method of producing aglycomodified protein, comprising

-   -   cleaving an oligosaccharide present on a first protein at a        GlcNAc-GlcNAc linkage to obtain a protein comprising a GlcNAc        residue having a reducing end;    -   attaching a modified sugar comprising a chemical handle to the        reducing end of said GlcNAc residue; and    -   mixing said first protein with a modified oligosaccharide having        a label capable of reacting with said chemical handle;    -   wherein said modified oligosaccharide attaches to the protein at        said chemical handle, thereby forming a glycomodified protein.

In another embodiment, said protein is an antibody. More particularly,the antibody is an IgG. In another embodiment, said oligosaccharide iscleaved using endoglycosidase H cleavage at the GlcNAc-GlcNAc linkage.In another embodiment, said modified sugar is attached to said reducingend using a mutant galactosyl transferase. In another embodiment, saidmutant is a Y289L mutant. In another embodiment, said modified sugar isan azide-modified sugar and said modified oligosaccharide is labeledwith alkyne. In another embodiment, said azide-modified sugar isUDP-GalNAz.

In another embodiment, said modified oligosaccharide is obtained by

-   -   cleaving an oligosaccharide present on a second protein at a        GlcNAc-GlcNAc linkage to obtain an oligosaccharide having a        GlcNAc residue having a reducing end;    -   labeling said oligosaccharide with a label capable of reacting        with said chemical handle.

In another embodiment, the reducing end of said cleaved oligosaccharideis treated with ammonium bicarbonate and said treated oligosaccharide isattached to an alkyne by a succinimidyl ester. In another embodiment,said second protein is synthesized in a different cell line or cell typethan said first protein. In another embodiment, said second protein issynthesized in a human cell.

In another embodiment, said modified oligosaccharide further comprises asecond label. In another embodiment, said second label is selected fromthe group consisting of therapeutic moieties, therapeutic agents,radioactive metal ions, DNA, protein, peptides, sugars, detectablelabels, biotin, and avidin.

Another aspect of the invention provides oligosaccharide-modifiedprotein obtained using the methods of any one of the embodiments.

Another aspect of the invention provides oligosaccharide-cleavedprotein, obtained using the method comprising treating a protein withendoglycosidase H. In another embodiment, said protein is an antibody.More particularly, said antibody is an IgG.

Another aspect of the invention provides an antibody comprising alabeled oligosaccharide. In another embodiment, said label is selectedfrom the group consisting of therapeutic moieties, therapeutic agents,radioactive metal ions, DNA, protein, peptides, sugars, detectablelabels, biotin, and avidin.

In another embodiment, the oligosaccharide is cleaved usingendoglycosidase M cleavage at the GlcNAc-GlcNAc linkage.

In another embodiment,

-   -   said cleavage of said first antibody is performed in the        presence of an OH-alkyne, and said endoglycosidase M attaches        said OH-alkyne to the reducing end of said cleaved GlcNAc        residue; and    -   said modified oligosaccharide is labeled with an azide residue.

In another embodiment, said oligosaccharide is cleaved from said secondantibody using endoglycosidase M cleavage at the GlcNAc-GlcNAc linkage.

In another embodiment,

-   -   said cleavage of said second antibody is performed in the        presence of an OH-alkyne, and said endoglycosidase M attaches        said OH-alkyne to the reducing end of said cleaved GlcNAc        residue, providing an alkyne-modified oligosaccharide; and    -   said chemical handle on said modified sugar on said first        antibody is an azide.

Another aspect of the invention provides an oligosaccharide-cleavedantibody, obtained using the method comprising treating an antibody withendoglycosidase M. In another embodiment, said antibody is an IgG.

Another aspect of the invention provides a method of labeling anantibody by labeling an oligosaccharide attached to said antibody,comprising incubating an antibody-producing cell in the presence of anunnatural sugar, wherein said unnatural sugar comprises a chemicalhandle.

In another embodiment, the antibody-producing cell is a recombinantcell. In another embodiment, said antibody-producing cell is ahybridoma. In another embodiment, said antibody is a IgG. In anotherembodiment, said chemical handle is selected from the group consistingof azides, triarylphosphines, or alkynes. In another embodiment, saidunnatural sugar is selected from the group consisting of GalNAz, ManNaz,and GlcNAz.

Another embodiment further comprises isolating said labeled antibodyfrom said antibody-producing cell.

Another embodiment, further comprises attaching a second label to saidisolated antibody, comprising contacting said isolated antibody with asecond label that is reactive with said chemical handle.

In another embodiment, said chemical handle is an azide and said secondlabel comprises or is modified to comprise an alkyne. In anotherembodiment, said second label is selected from the group consisting oftherapeutic moieties, therapeutic agents, radioactive metal ions, DNA,protein, peptides, sugars, detectable labels, biotin, and avidin.

Another aspect of the invention provides labeled antibody obtained usingthe methods of any of embodiments described herein.

Another aspect of the invention provides a kit, comprising

An unnatural sugar labeled with a chemical handle, and

A secondary label that will attach to said unnatural sugar at saidchemical handle.

In another embodiment, the kit further comprises instructions for invivo labeling of antibodies in antibody-producing cells. In anotherembodiment, said chemical handle is selected from the group consistingof azides, triarylphosphines, and alkynes. In another embodiment, saidunnatural sugar is selected from the group consisting of GalNAz, ManNAz,and GlcNAz. In another embodiment, said chemical handle is an azide andsaid secondary label comprises an alkyne.

The following examples describe specific aspects of the invention toillustrate the invention and to provide a description of the methods forthose of skill in the art. The examples should not be construed aslimiting the invention, as the examples merely provide specificmethodology useful in understanding and practicing the invention. Thereagents employed in the examples are commercially available or can beprepared using commercially available instrumentation, methods, orreagents known in the art. The foregoing examples illustrate variousaspects of the invention and practice of the methods of the invention.Each of the references cited in the examples is incorporated herein byreference in its entirety. The examples are not intended to provide anexhaustive description of the many different embodiments of theinvention nor to limit the selection of suitable reagents beyond whathas already been described above. Thus, although the forgoing inventionhas been described in some detail by way of illustration and example forpurposes of clarity of understanding, those of ordinary skill in the artwill realize readily that many changes and modifications can be madethereto without departing from the spirit or scope of the appendedclaims.

EXAMPLES Example 1 HPLC Verification of Monoclonal and PolyclonalAntibodies With and Without Oligosaccharide Cleavage by Endo HGlycosidase

Deglycosylation reaction: To 90 μL reduced and alkylated antibody (1mg/ml) add 40 μL 0.5M sodium citrate pH 5.5 and 10 μL EndoH_(f),(1,000,000 U/mL, New England BioLabs). Incubate for 48 hours at 37° C.with rocking. Samples were centrifuged to remove precipitate and theninjected directly onto the HPLC. Reversed-phase HPLC was performed on anAgilent Zorbax 300SB-CN column (4.6×150 mm, 3.5 μm) at 75° C. with aflow rate of 0.8 mL/min using a Waters 600E LC system. The mobile phaseincluded water with 0.1% TFA in solvent A and 80% n-propanol, 10%acetonitrile, 10% water with 0.1% TFA in solvent B (conditions of Rehderet al., J. Chrom. A, 1102 (2006) 164-175). Separation was accomplishedusing a linear gradient from 20 to 40% B over 30 mM Results are depictedin FIG. 1 and FIG. 2.

Example 2 Enzymatic Labeling of Chicken Anti-Goat Antibody With andWithout Oligosaccharide Cleavage by Endo H Gglycosidase

Chicken anti-goat IgG was washed into 50 mM MES pH 6.5 buffer on aVivaSpin 5000 MWCO filter column. For the deglycosylation reaction, analiquot of washed IgG at 1 mg/mL was incubated 24 hours at 37° C. in 50mM MES pH 6.5 containing 47.5 U/μL Endo H (New England BioLabs). Washed(undeglycosylated) or EndoH_(f) digested IgG was then azido-labeled withthe Click-iT O-GlcNAc enzymatic labeling kit using nondenaturingconditions: 0.5 mg/ml IgG in 50 mM MES pH 6.5, 120 mM NaCl, 11 mM MnCl₂,0.1 mM ZnCl₂, 50 μM UDP-GalNAz, 5 U/mL Antarctic phosphatase, and 0.65mg/mL GalT enzyme, overnight incubation at 4° C. The samples werepurified through P10 sizing resin packed into a 0.5 mL spin column into50 mM Tris pH 8. Collected fractions containing antibody were thenlabeled with the TAMRA Click-iT™ detection kit (C33370) and purifiedagain through P10 sizing resin packed into a 0.5 mL spin column into 50mM Tris pH 8 buffer. Approximately 250 ng was analyzed on a 4-12%BIS-TRIS gel using MOPS buffer. The gels were imaged on the BioRad FXimager using the 532 nm laser and 555 nm long pass emission filter (FIG.3A). The gels were post-stained with SYPRO® Ruby protein gel stain andimaged using the 488 nm laser and 555 nm long pass emission filter (FIG.3B).

Example 3 Metabolic Labeling of Antibodies With Azido Sugars

Mouse M96 hybridoma cells were fed the azido sugar analogues, Ac₄GalNAz,Ac₄ManNAz or Ac₄GlcNAz for five days. The cell supernatant (containingthe azido-labeled monoclonal antibody) was collected and proteins wereprecipitated in chloroform/methanol. Precipitated pellets wereresolubilized with 50 μL of 1% SDS, 100 mM TRIS pH 8, labeled with theTAMRA Click-iT™ detection kit (C33370) and 1 μg was analyzed on a 4-12%BIS-TRIS gel using MOPS buffer. The gels were imaged on the BioRad FXimager using the 532 nm laser and 555 nm long pass emission filter (FIG.4A). The gels were post-stained with SYPRO® Ruby protein gel stain andimaged using the 488 nm laser and 555 nm long pass emission filter (FIG.4B).

Example 4 Metabolic Labeling and “Click” Detection of GlycoproteinSubclasses

Jurkat cells were fed 40 μM Ac₄ManNAz or Ac₄GalNAz for 3 days (FIG. 5A)or 250 uM Ac₄GlcNAz overnight (FIG. 5B). Harvested cells were sonicatedin 50 mM Tris buffer, pH 8.0 with protease and phosphatase inhibitors,and the lysates were subjected to high-speed centrifugation (100K×g).The membrane pellet proteins from ManNAz- and GalNAz-treated cells, andthe soluble supernatant cells from the GlcNAc-treated cells, wereprecipitated with chloroform/methanol, dissolved in detergent, andlabeled with a fluorescent alkyne probe in the presence of 1 mM CuSO4,and 5 mM ascorbic acid (1). 10 μg of the labeled, precipitated proteinswere run on 1-D NuPAGE® Novex® 4-12% gels (Invitrogen). Images wereobtained on the Fuji FLA-3000 scanner (Fuji) using 532 nm excitation(FIG. 5A). Gels were then post stained with SYPRO® Ruby stain(Invitrogen) and imaged using excitation at 473 nm (FIG. 5B). Controllanes represent extracts from unfed cells but treated with thefluorescent probe. See FIGS. 5A-C.

Example 5 Separation of Ac₄GlcNAz-Treated Soluble Jurkat Cell Proteinsby 2-D Gels

Jurkat cells were cultured overnight with 250 uM Ac₄GlcNAz or DMSOvehicle (control unfed). Soluble lysate proteins were prepared as forExample 4 using sonication and ultracentrifugation and labeled for 1hour with a fluorescent alkyne probe. 40 μg of the labeled proteins wereprecipitated and resolubilized in 7M urea, 2M thiourea, 65 mM DTT, 2%CHAPS, 1% Zwittergent 3-10, 1% pH 3-10 carrier ampholytes and separatedon pH 3-10 IEF strips in the first dimension and 4-12% Bis-Tris gelswith MOPS buffer in the second dimension. Images were obtained on theFuji FLA-3000 scanner (Fuji) using 532 nm excitation Gels were then poststained with SYPRO® Ruby stain (Invitrogen) and imaged again usingexcitation at 473 nm. See FIG. 6.

Example 6 In Gel Detection of 40 and 50 kD Azide-Labeled Model Proteins

25 pmols each of 40 and 50 kD model proteins with single N-terminalazides were spiked into 100 μg of Jurkat cell lysates (upper panels), ornot (lower panels). Proteins were labeled with a fluorescent alkyneprobe, serially diluted as shown, and run on NuPAGE® Novex® 4-12% gels.Images (left panels) were obtained on the FLA-3000 scanner using 532 nmexcitation. Gels were then post stained with SYPRO® Ruby stain andimaged using excitation at 473 nm (right panels). Detection sensitivityof the labeled proteins is less than 10 femtomoles. See, FIG. 7.

Example 7 Labeling Efficiency of 40 and 50 kd Azide-Labeled ModelProteins is Unchanged in Complex Protein Extracts

Either 100 ng (25 pmol) or 10 ng (2.5 pmol) each of azide-labeled 40 Kd& 50 Kd proteins were labeled with fluorescent alkyne probe as above ina background of either 100, 50, 25, or 0 μg of control Jurkat lysate(left panel). Note: 100 μg of control lysate was added after labeling tothe ‘0 lysate’ to facilitate recovery of the labeled protein byprecipitation. The gel was post stained with SYPRO® Ruby total proteinstain. See FIG. 8.

Example 8 Enzymatic Labeling and Detection of α-crystallin O-GlcNAc

α-crystallin O-GlcNAc was enzymatically labeled with azide (UDP-GalNAz)using a modified b-GalT1 enzyme. The protein was subsequently reactedwith a fluorescent alkyne probe as described. The proteins were run on1-D NuPAGE® Novex® 4-12% gels at the dilutions shown. Note: Only 2-10%of α-crystallin is O-GlcNAc-modified and therefore the detectionsensitivity of the O-GlcNAc moiety is in the mid-to-low femtomole range(10-45 fmols). See FIGS. 9A-B.

Example 9 Comparison of GalT1 Enzyme Labeling with a-O-GlcNAc MonoclonalAntibody CTD 110.6

In FIG. 10A, α-crystallin O-GlcNAc was enzymatically labeled with themodified GalT1 enzyme and subsequently reacted with a biotin-alkyneprobe. The proteins were run on 1-D NuPAGE® Novex® 4-12% Bis-Tris gels,at the dilutions shown, and blotted onto PVDF membrane. The PVDFmembrane was then incubated in streptavidin-HRP and proteins weredetected using ECL Plus™ (GE Biosystems). Lane 2 (NE) represents the 8pmoL no-enzyme added control. Note: In FIG. 10A, the detectionsensitivity of O-GlcNAc by Western blot is in the low femtomole range(3-10 fmols). In FIG. 10B, untreated α-crystallin was run on 1-D gelsand blotted as described above. The PVDF membrane was processed usingthe O-GlcNAc Western Blot Detection Kit (Pierce) according tomanufacturer's instructions. The kit utilizes the CTD110.6 α-O-GlcNAcmonoclonal antibody. Lane 2 contains 5 ng of the positive control(O-GlcNAc-modified BSA) provided in the kit. No α-crystallin is detectedusing the antibody detection system. See FIGS. 10A and 10B.

Example 10 Multiplex Detection of O-GlcNAc Proteins, Phosphoproteins andTotal Proteins in the Same 2-D gel

Soluble extracts from Ac₄GlcNAz-fed Jurkat cells were labeled with UVexcitable alkyne dye for 2 hours. The chloroform/methanol precipitatedproteins were run on 2-D gels as described previously. The gel wasrinsed in water and imaged with UV transillumination and 600/bp emissionon a Lumi-Imager™ (Roche). The gel was then stained with Pro-Q® Diamondphosphoprotein stain, imaged with 532 nm excitation/580 LP emission on aFLA-3000 laser imager, stained with SYPRO® Ruby total protein stain, andimaged again with 473 nm excitation and 580 nm longpass emissionaccording to the manufacturer's instructions. See FIG. 11.

Example 11 Multiplexed Western Blot Detection of O-GlcNAc ModifiedProteins and Cofilin

25 μg of soluble Jurkat cell proteins were run on 2-D gels as describedand blotted onto PVDF membrane. The PVDF membrane was incubated inα-cofilin polyclonal Ab and detected with GAR-HRP secondary Ab with ECLPlus™ detection (GE Biosystems). After imaging, the blot was incubatedin streptavidin AP and O-GlcNAc proteins were detected using theWesternBreeze® chemiluminescent detection kit (Invitrogen). See FIG. 12.

Example 12 Differential Detection of O-GlcNAc Modified Proteins inControl and Inhibitor-Treated Cultured Cell Extracts

Jurkat cells cultured overnight with Ac₄GlcNAz with and without PUGNActreatment. PUGNAc is a commonly used inhibitor of O-GlcNAcase. SolubleJurkat lysate preparations were labeled with fluorescent alkyne. Lane 1)cells treated with 50 μM PUGNAc and 4 mM glucosamine 3 hrs prior toharvest; Lane 2) no treatment; Lanes 3-5) cells cultured overnight with250 μM Ac₄GlcNAz; Lanes 6-8) cells cultured overnight with 250 μMAc₄GlcNAz then treated with 50 μM PUGNAc and additional 250 μM Ac₄GlcNAz3 hrs prior to harvest. Proteins treated with PUGNAc (lanes 6-8) show amarked increase in O-GlcNAc staining over the untreated controls (lanes3-5). See FIG. 13.

Example 13 In-Gel Ligation of Glycoproteins

Fluorescent alkyne compounds for use in in-gel ligation are shown inFIGS. 18A-18D. Additionally, 2 potential fluorogenic alkynes are shownin FIGS. 18E and 18F. The TAMRA-alkyne compound, shown in the upperleft-hand frame, was used in in-gel staining experiments whereby azidogroups were incorporated into proteins in vitro using a reactiveazido-succinimidyl ester, or in vivo, by feeding cells azido-modifiedsugars.

Cell lysates were obtained from cultured Jurkat cells that were fedazido-modified sugars.

Azide-Alkyne Reaction Conditions for In-Gel or Western Blot Detection:

Component Volume Final Protein in 1% SDS, 50 mM Tris pH 8  50 uL 100-200ug Tris-HCl, pH 8.0 (1M) 7.5 uL 50 mM Propylene glycol  50 uL 25% CuSO₄(50 mM)   4 uL  1 mM DMSO, 0.5M   4 uL 10 mM H20 62.5 uL  to 200 uLAlkyne compound   2 uL 10 uM 1 mM (eg TAMRA or biotin) Na Ascorbate (100mM)  10 uL  5 mM

All components are combined, adding the ascorbate last and the solutionis vortexed gently, with a final solution volume of 200 uL. 10 uL of 100mM BCS is added (solution turns orange if CuI is present) and vortexedgently followed by a layer of argon. The solution is mixed on rotator atroom temperature for 1 hour.

After the reaction, the protein is precipitated out using the followingprotocol. 600 μL of MeOH is added to the reaction mixture and vortexed20 secs, (freeze 30 min if protein amount. is low), followed by 200 μLchloroform and vortexing 20 secs, then 450 μL H₂O vortex 20 secs. Thesolution is spun @18Kxg for 5 min. The upper phase is removed and 450 μLof MeOH is added followed by vortexing for 20 secs and spinning @18Kxgfor 5 min. The supernatant is removed and discarded. Finally, 600 μL ofMeOH is added, vortex, and briefly sonicate to disperse pellet, followedby spinning @18Kxg for 5 mM.

For sodium ascorbate dilution to 100 mM, dry sodium ascorbate (5 mg) iscombined with 250 μL H₂O.

FIG. 19, shows the results of electrophoresis of proteins labeled withthe TAMRA-Alkyne compound using the protocol provided. Lanes 2, 3, and 4on the left side of the gel represent cellular extracts that hadincorporated azide-modified sugars, lane 1 is the control, non-azidesugar fed cells. On the right, control azide labeled proteins (ovalbuminand myoglobin) (+) or non-labeled controls (−) are shown at varyingconcentrations. The results show very efficient and selective in-geldetection of azido-modified proteins.

Example 14

2.5 μg each of azido-ovalbumin and azido-myoglobin were spiked into 80ug of unlabeled Jurkat lysate. The lysate was then labeled with TAMRAalkyne for 2 hrs. The reaction contained 50 mM TRIS pH8, 25% propyleneglycol, 1 mM CuSO₄, 5 mM sodium ascorbate, 20 uM TAMRA alkyne. Thereactions were performed with and without a chelator (10 mM of eitherTPEN, EDTA, bathocuproine disulfonic acid (BCS) or neocuproine). Thecontrol reaction was performed without CuSO₄. After labeling, thesamples were precipitated, resolubilized in 7 mM urea/2 mM thiourea/65mM DTT/2% CHAPS/ and approximately 30 μg of each sample was analyzed on2-D gels (pH 4-7 IEF strips, 4-12% BIS-TRIS gels with MOPS buffer). TheTAMRA signal was imaged at 532 nm, excitation, 580 long pass emission ona Fuji FLA3000 then the gels were post-stained with SYPRO® Ruby totalprotein gel stain (FIG. 14 A). The results show that addition ofchelator greatly improves the resolution of the protein separation.bathocuproine disulfonic acid (BCS), a Cu I chelator, gives the bestresults. See total protein stain, FIG. 14 B.

In a second experiment, the samples and click labeling conditions werethe same, except that chelator treatments included the addition ofeither 5 mM TPEN, BCS, or Neocuproine at the beginning of the reaction.After labeling, the samples were precipitated, resolubilized in LDSbuffer +5 mM TCEP and serial 2-fold dilutions were performed. Dilutionswere loaded onto 4-12% BIS-TRIS gels with MOPS running buffer (250 ngeach of ovalbumin and myglobin in lane 1). FIG. 15 A shows that thechelators reduce the background of the image for the TAMRA signalwithout compromising sensitivity. In FIG. 15 B, post-staining withSypro® Ruby total protein gel stain shows that the band resolution ismuch better for the samples with chelator.

A further experiment testing the effect of chelators used the same clicklabeling conditions except that the chelator treatments includedaddition of either 7 mM, 5 mM, or 2 mM BCS; or 7 mM, 5 mM, or 2 mMneocuproine. The lanes marked with an asterisk in FIG. 16A indicatereactions in which the CuSO4 and BCS were added to the reaction andvortexed prior adding the sodium ascorbate. In all other reactions theCuSO4 and sodium ascorbate were added and vortexed prior to adding theBCS. The gels show that it is imperative to add the sodium ascorbate andCuSO4 to the reaction tube and mix prior to adding the chelator. If thechelator and CuSO4 are added and vortexed prior to adding the sodiumascorbate, the azide-alkyne labeling does not proceed, suggesting thatthe chelator inhibits the reduction of Cu (II) to Cu (I).

Example 15 Enzymatic Labeling of Antibodies Using Click Chemistry

Goat IgG antibodies were reduced and alkylated, then deglycosylated in 2separate aliquots using Endo H enzyme. Deglycosylated antibodies (2separate preps) were then labeled with GalNAz using 33 ng/uL Gal T1Y289L enzyme and 500 uM UDP GalNAz (0.5 ug/uL goat antibody) in a 150 uLreaction. Reactions were incubated at 4 degrees C. overnight. 4-500 ngof goat antibody (treated as listed on gel; either no-GalNAz control orazide labeled) was loaded into each lane of a 4-12% Bis Tris gel.Electrophoresis was performed at 200v for ˜50 min using MES buffer. Gelswere stained with TAMRA-alkyne stain and imaged on the Fuji imager at532 nm (excitation) and 580 nm emission. Gels were poststained withSYPRO Ruby using the overnight protocol. See FIGS. 17A and 17B.

U.S. Patent applications with attorney docket numbers IVGN 745 and IVGN745.1 all filed on Feb. 12, 2007, claiming priority to U.S. ProvisionalApplication Nos. 60/772,221 and 60/804,640 are hereby incorporated byreference.

The entirety of each patent, patent application, publication anddocument referenced herein hereby is incorporated by reference in theirentirety, including all tables, drawings, and figures. All patents andpublications are herein incorporated by reference to the same extent asif each was specifically and individually indicated to be incorporatedby reference. Citation of the above patents, patent applications,publications and documents is not an admission that any of the foregoingis pertinent prior art, nor does it constitute any admission as to thecontents or date of these publications or documents.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andsystems similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the methods, devices,and materials are now described.

Modifications may be made to the foregoing without departing from thescope, spirit and basic aspects of the invention. Although the inventionhas been described in substantial detail with reference to one or morespecific embodiments, those of ordinary skill in the art will recognizethat changes may be made to the embodiments specifically disclosed inthis application, and yet these modifications and improvements arewithin the scope and spirit of the invention. One skilled in the artreadily appreciates that the present invention is well adapted to carryout the objects and obtain the ends and advantages mentioned, as well asthose inherent therein. The examples provided herein are representativeof specific embodiments, are exemplary, and are not intended aslimitations on the scope of the invention.

The invention illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, the terms and expressions which have been employed are used asterms of description and not of limitation, equivalents of the featuresshown and described, or portions thereof, are not excluded, and it isrecognized that various modifications are possible within the scope ofthe invention. Embodiments of the invention are set forth in thefollowing claims.

1. A method of producing a glycomodified protein, comprising attaching amodified sugar comprising a chemical handle to a GlcNAc residue on afirst protein; and mixing said first protein with a reporter molecule,carrier molecule or solid support capable of reacting with said chemicalhandle; wherein said reporter molecule, carrier molecule or solidsupport attaches to the protein at said chemical handle, thereby forminga glycomodified protein. 2-20. (canceled)
 21. A method of labeling anprotein by labeling an oligosaccharide attached to said protein,comprising incubating an protein-producing cell in the presence of anunnatural sugar, wherein said unnatural sugar comprises a chemicalhandle.
 22. An antibody comprising a labeled oligosaccharide.
 23. Theantibody of claim 22, wherein the labeled oligosaccharide comprises anazido-modified sugar.
 24. The antibody of claim 23, wherein the labeledoligosaccharide comprises a reporter molecule, a carrier molecule or asolid support capable of reacting with said azido-modified sugar. 25.The antibody of claim 22, wherein the antibody is humanized.
 26. Theantibody of claim 22, wherein the antibody is a therapeutic antibody.27. The antibody of claim 24, wherein the reporter molecule is afluorescent dye, enzyme, radiolabel, a metal chelator, or a detectablesubstrate.
 28. The antibody of claim 22, wherein the label is selectedfrom the group consisting of therapeutic moieties, therapeutic agents,radioactive metal ions, DNA, protein, peptides, sugars, detectablelabels, biotin, and avidin.
 29. The antibody of claim 22, wherein theantibody is obtained using a method comprising: attaching a modifiedsugar comprising a chemical handle to a GlcNAc residue on the antibody;and mixing said antibody with a reporter molecule, carrier molecule orsolid support capable of reacting with said chemical handle; whereinsaid reporter molecule, carrier molecule or solid support attaches tothe antibody at said chemical handle, thereby forming the antibodycomprising the labeled oligosaccharide.